US 200901 63434A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0163434 A1 BADER et al. (43) Pub. Date: Jun. 25, 2009

(54) MIR-20 REGULATED AND Related U.S. Application Data PATHWAYS AS TARGETS FOR THERAPEUTIC INTERVENTION (60) Provisional application No. 60/915,026, filed on Apr. 30, 2007. (76) Inventors: Andreas G. BADER, Austin, TX Publication Classification (US); Mike BYROM, Austin, TX (US); Charles D. JOHNSON, (51) Int. Cl. Austin, TX (US); David BROWN, A6IR 48/00 (2006.01) Austin, TX (US) CI2O I/68 (2006.01) A6IP 43/00 (2006.01) Correspondence Address: (52) U.S. Cl...... 514/44; 435/6; 977/773 Fulbright & Jaworski L.L.P. 600 Congress Avenue, Suite 2400 (57) ABSTRACT Austin, TX 78701 (US) The present invention concerns methods and compositions for identifying genes or genetic pathways modulated by miR (21) Appl. No.: 12/112,291 20a, using miR-20a to modulate a or gene pathway, using this profile in assessing the condition of a patient and/or (22) Filed: Apr. 30, 2008 treating the patient with an appropriate miRNA. US 2009/0163434 A1 Jun. 25, 2009

MR-2O REGULATED GENES AND cers (reviewed in Esquela-Kerscher and Slack, 2006; Calin PATHWAYS AS TARGETS FOR and Croce, 2006). Differential expression of almost all miR THERAPEUTIC INTERVENTION NAS across numerous cancer types has been observed (Lu et al., 2005). Most such studies link miRNAs to cancer only by indirect evidence. However. He et al. (2005a) has provided 0001. This application claims priority to U.S. Provisional more direct evidence that miRNAs may contribute directly to Patent application Ser. No. 60/915,026 filed Apr. 30, 2007, causing cancer, by forcing the over-expression of six miR which is incorporated herein by reference in its entirety. NAS in mice, including miR-20a, that resulted in a significant increase in B cell lymphomas. BACKGROUND OF THE INVENTION 0009. The inventors previously demonstrated that hsa 0002 I. Field of the Invention miR-20a is involved with the regulation of numerous cell 0003. The present invention relates to the fields of molecu activities that represent intervention points for cancer therapy lar biology and medicine. More specifically, the invention and for therapy of other diseases and disorders (U.S. patent relates to methods and compositions for the treatment of application Ser. No. 1 1/141,707 filed May 31, 2005 and Ser. diseases or conditions that are affected by miR-20 microR No. 1 1/273,640 filed Nov. 14, 2005, both of which are incor NAS, microRNA expression, and genes and cellular pathways porated by reference). Over-expression of miR-20a signifi directly and indirectly modulated by such. cantly reduced viability of Jurkat cells, a human T-cell line 0004 II. Background derived from leukemic peripheral blood, while significantly 0005. In 2001, several groups used a cloning method to increasing the viability and proliferation of primary normal isolate and identify a large group of “microRNAs (miRNAs) human T-cells. Cell regulators that enhance viability of nor from C. elegans, Drosophila, and humans (Lagos-Quintana et mal cells while decreasing viability of cancerous cells repre al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Several sent useful therapeutic treatments for cancer. Hsa-miR-20a hundred miRNAs have been identified in plants and ani increased apoptosis (induced death of cells with oncogenic mals—including humans—that do not appear to have endog potential) in A549 lung cancer cells and increased the per enous siRNAs. Thus, while similar to siRNAs, miRNAs are centage of BJ cells (human foreskin primary cells) in the S distinct. phase of the cell cycle while reducing the percentage of those 0006 miRNAs thus far observed have been approximately cells in the G1 phase of the cell cycle. The inventors observed 21-22 nucleotides in length, and they arise from longer pre that expression of hsa-miR-20a is higher in white blood cells cursors transcribed from non--encoding genes. See from patients with chronic lymphocytic leukemia than in the review of Carrington et al. (2003). The precursors form struc same cells from normal patients. Others have shown that tures that fold back on themselves in self-complementary hsa-miR-20a regulates the translational yield of the transcrip regions; they are then processed by the nuclease Dicer (in tion factor, E2F1 (O’Donnell et al., 2005) and appears to be animals) or DCL1 (in plants) to generate the short double over-expressed in colon, pancreas, and prostate tumors while stranded miRNA. One of the miRNA strands is incorporated being down-regulated in breast cancer tumors (Volinia et al., into a complex of and miRNA called the RNA 2006). induced silencing complex (RISC). The miRNA guides the 0010 Bioinformatics analyses Suggest that any given RISC complex to a target mRNA, which is then cleaved or miRNA may bind to and alter the expression of up to several translationally silenced, depending on the degree of sequence hundred different genes. In addition, a single gene may be complementarity of the miRNA to its target mRNA. Cur regulated by several miRNAs. Thus, each miRNA may regu rently, it is believed that perfect or nearly perfect complemen late a complex interaction among genes, gene pathways, and tarity leads to mRNA degradation, as is most commonly gene networks. Mis-regulation or alteration of these regula observed in plants. In contrast, imperfect base pairing, as is tory pathways and networks, involving miRNAS, are likely to primarily found in animals, leads to translational silencing. contribute to the development of disorders and diseases such However, recent data Suggest additional complexity (Bagga as cancer. Although bioinformatics tools are helpful in pre et al., 2005; Lim et al., 2005), and mechanisms of gene dicting miRNA binding targets, all have limitations. Because silencing by miRNAS remain under intense study. of the imperfect complementarity with their target binding 0007. Many miRNAs are conserved among diverse organ sites, it is difficult to accurately predict the mRNA targets of isms, and this has led to the Suggestion that miRNAS are miRNAs with bioinformatics tools alone. Furthermore, the involved in essential biological processes throughout the life complicated interactive regulatory networks among miRNAS span of an organism (Esquela-Kerscher and Slack, 2006). In and target genes make it difficult to accurately predict which particular, miRNAS have been implicated in regulating cell genes will actually be mis-regulated in response to a given growth and cell and tissue differentiation—cellular processes miRNA. that are associated with the development of cancer. For 0011 Correcting errors or modulating instance, lin-4 and let-7 both regulate passage from one larval gene expression by manipulating miRNA expression or by state to another during C. elegans development (Ambros, repairing miRNA mis-regulation represent promising meth 2001). mir-14 and bantam are Drosophila miRNAs that regu ods to repair genetic disorders and cure diseases like cancer. late cell death, apparently by regulating the expression of A current, disabling limitation of this approach is that, as genes involved in apoptosis (Brennecke et al., 2003, Xu et al., mentioned above, the details of the regulatory pathways and 2003). networks that are affected by any given miRNA remain gen 0008 Research on microRNAs is increasing as scientists erally unidentified. Besides E2F1, the genes, gene pathways, are beginning to appreciate the broad role that these mol and gene networks that are regulated by miR-20 in cancerous ecules play in the regulation of eukaryotic gene expression. In cells remain largely unknown. Currently, this represents a particular, several recent studies have shown that expression significant limitation for treatment of cancers in which miR levels of numerous miRNAs are associated with various can 20 may play a role. A need exists to identify the genes, genetic US 2009/0163434 A1 Jun. 25, 2009 pathways, and genetic networks that are regulated by or that tissues or subjects would resultina therapeutic response. The may regulate hsa-miR-20 expression. identities of key genes that are regulated directly or indirectly by miR-20 and the disease with which they are associated are SUMMARY OF THE INVENTION provided herein. In certain aspects a cell may be an epithelial, 0012. The present invention provides additional composi stromal, or mucosal cell. The cell can be, but is not limited to tions and methods by identifying genes that are direct targets brain, a neuronal, a blood, an esophageal, a lung, a cardiovas for miR-20 regulation or that are indirect or downstream cular, a liver, a breast, a bone, a thyroid, a glandular, an targets of regulation following the miR-20-mediated modifi adrenal, a pancreatic, a stomach, a intestinal, a kidney, a cation of another gene(s) expression. Furthermore, the inven bladder, a prostate, a uterus, an ovarian, a testicular, a splenic, tion describes gene, disease, and/or physiologic pathways askin, a smooth muscle, a cardiac muscle, or a striated muscle and networks that are influenced by miR-20 and its family cell. In certain aspects, the cell, tissue, or target may not be members. In certain aspects, compositions of the invention defective in miRNA expression yet may still respond thera are administered to a Subject having, Suspected of having, or peutically to expression or over expression of an miRNA. at risk of developing a metabolic, an immunologic, an infec miR-20 could be used as a therapeutic target for any of these tious, a cardiovascular, a digestive, an endocrine, an ocular, a diseases. genitourinary, a blood, a musculoskeletal, a nervous system, 0016. In certain aspects, the cell, tissue, or target may not a congenital, a respiratory, a skin, or a cancerous disease or be defective in miRNA expression yet may still respond condition. therapeutically to expression or over expression of a miRNA. 0013. In particular aspects, a subject or patient may be miR-20 could be used as a therapeutic target for any of these selected for treatment based on expression and/or aberrant diseases or conditions. In certain embodiments miR-20 or its expression of one or more miRNA or mRNA. In a further compliment can be used to modulate the activity of miR-20 or aspect, a subject or patient may be selected for treatment a miR-20 regulated gene in a subject, organ, tissue, or cell. based on aberrations in one or more biologic or physiologic 0017. A cell, tissue, or subject may be a cancer cell, a pathway(s), including aberrant expression of one or more cancerous tissue, harbor cancerous tissue, or be a Subject or gene associated with a pathway, or the aberrant expression of patient diagnosed or at risk of developing a disease or condi one or more protein encoded by one or more gene associated tion. In certain aspects a cancer cell is a neuronal, glial, lung, with a pathway. In still a further aspect, a Subject or patient liver, brain, breast, bladder, blood, leukemic, colon, endome may be selected based on aberrations in both miRNA expres trial, stomach, skin, ovarian, fat, bone, cervical, esophageal, sion, or biologic or physiologic pathway(s). A subject may be pancreatic, prostate, kidney, or thyroid cell. In still a further assessed for sensitivity, resistance, and/or efficacy of a aspect cancer includes, but is not limited to astrocytoma, therapy or treatment regime based on the evaluation and/or acute myelogenous leukemia, breast carcinoma, bladder car analysis of miRNA or mRNA expression or lack thereof. A cinoma, cervical carcinoma, colorectal carcinoma, endome subject may be evaluated for amenability to certain therapy trial carcinoma, esophageal squamous cell carcinoma, prior to, during, or after administration of one or therapy to a glioma, glioblastoma, gastric carcinoma, hepatocellular car Subject or patient. Typically, evaluation or assessment may be cinoma, Hodgkin lymphoma, leukemia, lipoma, melanoma, done by analysis of miRNA and/or mRNA, as well as com mantle cell lymphoma, myxofibrosarcoma, multiple bination of other assessment methods that include but are not myeloma, neuroblastoma, non-Hodgkin lymphoma, lung limited to histology, immunohistochemistry, blood work, etc. carcinoma, non-Small cell lung carcinoma, ovarian carci 0014. In some embodiments, an infectious disease or con noma, esophageal carcinoma, osteosarcoma, pancreatic car dition includes a bacterial, viral, parasite, or fungal infection. cinoma, prostate carcinoma, squamous cell carcinoma of the Many of these genes and pathways are associated with vari head and neck, thyroid carcinoma, urothelial carcinoma. ous cancers and other diseases. Cancerous conditions 0018 Embodiments of the invention include methods of include, but are not limited to astrocytoma, acute myelog modulating gene expression, or biologic or physiologic path enous leukemia, breast carcinoma, bladder carcinoma, cervi ways in a cell, a tissue, or a subject comprising administering cal carcinoma, colorectal carcinoma, endometrial carcinoma, to the cell, tissue, or Subject an amount of an isolated nucleic esophageal squamous cell carcinoma, glioma, glioblastoma, acid or mimetic thereof comprising a miR-20 nucleic acid gastric carcinoma, hepatocellular carcinoma, Hodgkin lym sequence in an amount Sufficient to modulate the expression phoma, leukemia, lipoma, melanoma, mantle cell lymphoma, of a gene or genes modulated by a miR-20 miRNA. A "miR myxofibrosarcoma, multiple myeloma, neuroblastoma, non 20 nucleic acid sequence' includes the full length precursor Hodgkin lymphoma, lung carcinoma, non-Small cell lung or processed (i.e., mature) sequence of miR-20 and related carcinoma, ovarian carcinoma, esophageal carcinoma, sequences set forth herein, as well as 5, 6, 7, 8, 9, 10, 11, 12. osteosarcoma, pancreatic carcinoma, prostate carcinoma, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 squamous cell carcinoma of the head and neck, thyroid car or more nucleotides of the precursor miRNA or its processed cinoma, urothelial carcinoma wherein the modulation of one sequence, including all ranges and integers there between. In or more gene is sufficient for a therapeutic response. Typi certain embodiments, the miR-20 nucleic acid sequence con cally a cancerous condition is an aberrant hyperproliferative tains the full-length processed miRNA sequence and is condition associated with the uncontrolled growth or inability referred to as a “miR-20 full-length processed nucleic acid to undergo cell death, including apoptosis. sequence.” In still further aspects, the miR-20 nucleic acid 0015 The altered expression or function of miR-20 in comprises at least a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. cells would lead to changes in the expression of these key 18, 19, 20, 21, 22, 23, 24, 25, or 50 nucleotide (including all genes and contribute to the development of disease or other ranges and integers there between) segment of miR-20 that is conditions. Introducing miR-20 (for diseases where the at least 75,80, 85,90, 95, 98.99 or 100% identical to SEQID miRNA is down-regulated) or a miR-20 inhibitor (for dis NO:1 to SEQID NO:269. In certain aspects, a subset of these eases where the miRNA is up-regulated) into disease cells or miRNAs will be used that include somebut not all of the listed US 2009/0163434 A1 Jun. 25, 2009

miR-20 family members. It is contemplated that one or more MAT0001142 SEQ ID NO:84); gga-miR-17-3p (MI miR-20 family members or miR-20 miRNAs may be specifi MAT0001115 SEQ ID NO:85); gga-miR-17-5p cally excluded from certain embodiments of the invention. (MIMAT0001114 SEQ ID NO:86); gga-miR-18a (MI For instance, in one embodiment only sequences comprising MAT0001113 SEQ ID NO:87); gga-miR-18b (MI the consensus sequence of SEQID NO:269 will be included MAT0001141 SEQ ID NO:88): ggo-miR-106a (MI with all other miRNAs excluded. The general term miR-20 MAT0002795 SEQ ID NO:89); go-miR-106b includes all members of the miR-20 family. The mature (MIMAT0002758 SEQ ID NO:90); ggo-miR-17-3p (MI sequences of miR-20 family includes hsa-miR-20a (MI MAT0002659 SEQ ID NO:91); go-miR-17-5p (MI MAT0000075, SEQ ID NO:1); hsa-miR-20b (MI MAT0002658 SEQ ID NO:92); go-miR-18 (MI MAT0001413, SEQ ID NO:2); age-miR-20 (MI MAT0002660 SEQ ID NO:93); ggo-miR-93 MAT0002676, SEQ ID NO:3); bta-miR-20a (MIMAT0002759 SEQ ID NO:94); hsa-miR-106a (MI (MIMAT0003527, SEQ ID NO:4); bta-miR-20b (MI MAT0000103 SEQ ID NO:95); hsa-miR-106b (MI MAT0003796, SEQ ID NO:5); dre-miR-20a (MI MAT0000680 SEQ ID NO:96); hsa-miR-17-3p (MI MAT0001786, SEQ ID NO:6); dre-miR-20a (MI MAT0000071 SEQ ID NO:97); hsa-miR-17-5p MAT0003400, SEQ ID NO:7); dre-miR-20b (MIMAT0000070 SEQ ID NO:98); hsa-miR-18a (MI (MIMAT0001778, SEQ ID NO:8); fru-miR-20 (MI MAT0000072 SEQ ID NO:99); hsa-miR-18a (MI MAT0003083, SEQ ID NO:9); gga-miR-20a (MI MAT0002891 SEQ ID NO:100); hsa-miR-18b (MI MAT0001111, SEQ ID NO:10); gga-miR-20b (MI MAT0001412 SEQ ID NO:101); hsa-miR-93 MAT0001411, SEQ ID NO:11); ggo-miR-20 (MIMAT0000093 SEQ ID NO:102); lca-miR-17-3p (MI (MIMAT0002662, SEQ ID NO:12); lca-miR-20 (MI MAT0002666 SEQ ID NO:103): 1ca-miR-17-5p (MI MAT0002669, SEQ ID NO:13): lla-miR-20 (MI MAT0002665 SEQ ID NO: 104); lca-miR-18 (MI MAT0002718, SEQ ID NO:14); mdo-miR-20 (MI MAT0002667 SEQ ID NO:105): lla-miR-106b MAT0004169, SEQ ID NO:15); mml-miR-20 (MIMAT0002777 SEQ ID NO: 106): lla-miR-17-3p (MI (MIMAT0002704, SEQ ID NO:16); mmu-miR-20a (MI MAT0002715 SEQ ID NO:107): lla-miR-17-5p (MI MAT0000529, SEQ ID NO:17); mmu-miR-20b (MI MAT0002714 SEQ ID NO:108): lla-miR-18 (MI MAT0003187, SEQ ID NO:18): mne-miR-20 (MI MAT0002716 SEQ ID NO:109): lla-miR-93 MAT0002725, SEQ ID NO:19); ppa-miR-20 (MIMAT0002778 SEQ ID NO:110); mdo-miR-17-3p (MI (MIMAT0002683, SEQ ID NO:20): ppy-miR-20 (MI MAT0004166 SEQ ID NO:111); mdo-miR-17-5p (MI MAT0002690, SEQ ID NO:21); ptr-miR-20 (MI MAT0004165 SEQ ID NO:112); mdo-miR-18 (MI MAT0002697, SEQ ID NO:22): rno-miR-20a (MI MAT0004167 SEQ ID NO:113): mdo-miR-93 MAT0000602, SEQ ID NO:23): rno-miR-20a: (MIMAT0004178 SEQ ID NO:114); mml-miR-106a (MI (MIMAT0000603, SEQ ID NO:24); rno-miR-20b (MI MAT0002798 SEQ ID NO:115); mml-miR-106b (MI MAT0003211, SEQ ID NO:25): rno-miR-20b* (MI MAT0002772 SEQ ID NO: 116); mml-miR-17-3p (MI MAT0003212, SEQ ID NO:26); sla-miR-20 (MI MAT0002701 SEQ ID NO:117); mml-miR-17-5p MAT0002711, SEQ ID NO:27); ssc-miR-20 (MIMAT0002700 SEQ ID NO:118); mml-miR-18 (MI (MIMAT0002129, SEQ ID NO:28); tini-miR-20 (MI MAT0002702 SEQ ID NO: 119); mml-miR-93 (MI MAT0003084, SEQ ID NO:29): xla-miR-20 (MI MAT0002773 SEQ ID NO:120); mmu-miR-106a (MI MAT0001348, SEQ ID NO:30); Xtr-miR-20a (MI MAT0000385 SEQ ID NO:121); immu-miR-106b MAT0003669, SEQ ID NO:31): Xtr-miR-20a: (MIMAT0000386 SEQ ID NO:122); immu-miR-17-3p (MI (MIMAT0003670, SEQ ID NO:32); and/or Xtr-miR-20b MAT0000650 SEQ ID NO:123); immu-miR-17-5p (MI (MIMAT0003707, SEQID NO:33). MAT0000649 SEQ ID NO.124); mmu-miR-18 (MI 0019. Other members of the miR-20 family, as designated MAT0000528 SEQ ID NO:125); mmu-miR-93 by the Sanger database, include age-miR-106a (MI (MIMAT0000540 SEQ ID NO:126); mne-miR-106a (MI MAT0002796, SEQ ID NO:63); age-miR-106b (MI MAT0002802 SEQ ID NO.127); mne-miR-106b (MI MAT0002761 SEQ ID NO:64); age-miR-17-3p (MI MAT0002780 SEQ ID NO:128); mne-miR-17-3p (MI MAT0002673 SEQ ID NO:65); age-miR-17-5p MAT0002722 SEQ ID NO:129); mne-miR-17-5p (MIMAT0002672 SEQ ID NO:66); age-miR-18 (MI (MIMAT0002721 SEQ ID NO: 130); mne-miR-18 (MI MAT0002674 SEQ ID NO:67); age-miR-93 (MI MAT0002723 SEQ ID NO:131); mne-miR-93 (MI MAT0002762 SEQ ID NO:68); bta-miR-106 (MI MAT0002781 SEQ ID NO:132); ppa-miR-106a (MI MAT0003784 SEQ ID NO:69); bta-miR-17-3p MAT0002797 SEQ ID NO:133): ppa-miR-106b (MIMAT0003816 SEQ ID NO:70); bta-miR-17-5p (MI (MIMAT0002763 SEQ ID NO:134); ppa-miR-17-3p (MI MAT0003815 SEQ ID NO:71); bta-miR-18a (MI MAT0002680 SEQ ID NO:135): ppa-miR-17-5p (MI MAT0003526 SEQ ID NO:72); bta-miR-18b (MI MAT0002679 SEQ ID NO:136); ppa-miR-18 (MI MATOOO3517 SEQ ID NO:73); bta-miR-93 MAT0002681 SEQ ID NO:137); ppa-miR-93 (MIMAT0003837 SEQ ID NO:74); dre-miR-17a (MI (MIMAT0002764 SEQ ID NO: 138): ppy-miR-106a (MI MAT0001777 SEQ ID NO:75); dre-miR-17a: (MI MAT0002799 SEQ ID NO:139); ppy-miR-106b (MI MAT0003396 SEQ ID NO:76); dre-miR-18a (MI MAT0002766 SEQ ID NO:140); ppy-miR-17-3p (MI MAT0001779 SEQ ID NO:77); dre-miR-18b MAT0002687 SEQ ID NO:141); ppy-miR-17-5p (MIMAT0001780 SEQ ID NO:78); dre-miR-18b (MI (MIMAT0002686 SEQ ID NO: 142); ppy-miR-18 (MI MAT0003397 SEQ ID NO:79); dre-miR-18c (MI MAT0002688 SEQ ID NO:143): ppy-miR-93 (MI MAT0001781 SEQ ID NO:80); dre-miR-93 (MI MAT0002767 SEQ ID NO: 144); ptr-miR-106a (MI MATOOO1810 SEQ ID NO:81); fru-miR-17 MAT0002800 SEQ ID NO:145); ptr-miR-106b (MIMAT0002916 SEQ ID NO:82); fru-miR-18 (MI (MIMAT0002769 SEQ ID NO:146); ptr-miR-17-3p (MI MAT0002918 SEQ ID NO:83); gga-miR-106 (MI MAT0002694 SEQ ID NO:147); ptr-miR-17-5p (MI

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lar the miR-20 family members designated as miR-20s com tified in Tables 1,3,4, and 5. Modulation includes modulating prises a consensus of YAAAGUGCUYAYAGUGCAGGU transcription, mRNA levels, mRNA translation, and/or pro SEQID NO:269. tein levels in a cell, tissue, or organ. In certain aspects the 0022. In specific embodiments, a miR-20 containing expression of a gene or level of a gene product, such as nucleic acid or a miR-20 nucleic acid is hsa-miR-20a and/or mRNA, is down-regulated or up-regulated. In a particular hsa-miR-20b, or a variations thereof. In certain aspects miR aspect the gene modulated comprises or is selected from (and 20 is miR-20a or miR-20b. miR-20 can behsa-mir-20, includ may even exclude) 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, inghsa-miR-20a or hsa-miR20b. Inafurther aspect, a miR-20 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26. 27, 28, or all of the nucleic acid can be administered with 1, 2, 3, 4, 5, 6, 7, 8, 9, genes identified in Tables 1, 3, 4, and 5, or any combinations 10 or more miRNAS. miRNA can be administer concurrently, thereof. In certain embodiments a gene modulated or selected in sequence or in an ordered progression. In certain aspects to be modulated is from Table 1. In further embodiments a miR-20 can be administered in combination with one or more gene modulated or selected to be modulated is from Table 3. of let-7, miR-15a, miR-16, miR-21, miR-26a, miR-31, miR In still further embodiments a gene modulated or selected to 34a, miR-126, miR-143, miR-145, miR-147, miR-188, miR be modulated is from Table 4. In yet further embodiments a 200b, miR-200c, miR-215, miR-216, miR-292-3p, and/or gene modulated or selected to be modulated is from Table 5. miR-331. All or combinations of miRNAs may be adminis Embodiments of the invention may also include obtaining or tered in a single formulation. Administration may be before, assessing a gene expression profile or miRNA profile of a during or after a second therapy. target cell prior to selecting the mode of treatment, e.g., 0023 miR-20 nucleic acids may also include various het administration of a miR-20 nucleic acid or mimetic. The erologous nucleic acid sequences, i.e., those sequences not database content related to nucleic acids and genes desig typically found operatively coupled with miR-20 in nature, nated by an accession number or a database Submission are such as promoters, enhancers, and the like. The miR-20 incorporated herein by reference as of the filing date of this nucleic acid can be a recombinant nucleic acid, and can be a application. In certain aspects of the invention one or more ribonucleic acid or a deoxyribonucleic acid. The recombinant miRNA may modulate a single gene. In a further aspect, one nucleic acid may comprise a miR-20 expression cassette, i.e., or more genes in one or more genetic, cellular, or physiologic a nucleic acid segment that expresses a nucleic acid when pathways can be modulated by one or more miRNAs, includ introduce into an environment containing components for ing miR-20 nucleic acids in combination with other miRNAs. nucleic acid synthesis. In a further aspect, the expression cassette is comprised in a viral vector, or plasmid DNA vector TABLE 1 or other therapeutic nucleic acid vector or delivery vehicle, Genes with increased (positive values) or decreased (negative values) including liposomes and the like. In certain aspects, viral expression following transfection of human cancer cells with vectors can be administered at 1x10, 1x10, 1x10" 1x10, pre-miRhsa-miR-20a. 1x10, 1x107, 1x10, 1x10, 1x10, 1x10'', 1x10, 1x10", 1x10" pfu or viral particle (vp). Gene Symbol RefSeq, Transcript ID (Pruitt et al., 2005) A log2 0024. In a particular aspect, the miR-20 nucleic acid is a ABCA1 NM OO55O2 -101473 synthetic nucleic acid. Moreover, nucleic acids of the inven ALDH6A1 NM OO5589 O4418 tion may be fully or partially synthetic. In still further aspects, ANG .. NM OO1145 NM OO2937 O.8315O1 RLNASE4 NM 194430 NM 194431 a nucleic acid of the invention or a DNA encoding such can be ANK3 NM OO1149 NM 020987 16621 administered at 0.001, 0.01, 0.1, 1, 10, 20, 30, 40, 50, 100, ANKRD46 NM 1984O1 0.746,793 200, 400, 600, 800, 1000, 2000, to 4000 ug or mg, including ANTXR1 NM 018153 NM O32208 -113558 all values and ranges there between. In yet a further aspect, NM 053034 APOEH NM 000042 21612 nucleic acids of the invention, including synthetic nucleic AQP3 NM 004925 23947 acid, can be administered at 0.001, 0.01, 0.1, 1, 10, 20, 30, 40, ARG2 NM 001172 2.10966 50, 100, to 200 ug or mg per kilogram (kg) of body weight. ARIDSB NM 032199 35503 Each of the amounts described herein may be administered ARL7 NM 005.737 -1.06672 ARTS-1 NM 016442 -1.08712 over a period of time, including 0.5, 1,2,3,4,5,6,7,8,9, 10. ATP6VOE NM OO3945 -10247 minutes, hours, days, weeks, months or years, including all ATP9A NM OO6045 O1985 values and ranges there between. AXL NM OO1699 NM O21913 O.763332 BCL2A1 NM OO4049 -1.77411 0025. In certain embodiments, administration of the com BEAN XM 375.359 -0.714992 position(s) can be enteral or parenteral. In certain aspects, BICD2 NM OO10O3800 NM O15250 -0.781.188 enteral administration is oral. In further aspects, parenteral BTG3 NM OO6806 -1.192SS administration is intralesional, intravascular, intracranial, BTN3A2 NM 007047 -O.765137 C190rf2 NM 003796 NM 134447 -O.7SS164 intrapleural, intratumoral, intraperitoneal, intramuscular, C21orf25 NM 199050 -O.79.1738 intralymphatic, intraglandular, Subcutaneous, topical, intra C2Orf17 NM O24293 -O.94.5852 bronchial, intratracheal, intranasal, inhaled, or instilled. C2Orf31 O.942376 Compositions of the invention may be administered region CSOrf13 NM 004772 O.909743 ally or locally and not necessarily directly into a lesion. C6orf120 NM 001029863 -0.719609 C6orf216 NM 206908 NM 206910 O.743816 0026. In certain aspects, the gene or genes modulated NM 206911 NM 206912 comprises 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 20, 25, XR OOO259 CA12 NM OO1218 NM 206925 -O.88S975 30, 35, 40, 45, 50, 100, 150, 200 or more genes or combina CCL2 NM OO2982 -12O227 tions of genes identified in Tables 1, 3, 4, and 5. In still further CCND1 NM 053056 -1.21374 aspects, the gene or genes modulated may exclude 1, 2, 3, 4, CCNG1 NM 004060 NM 199246 O.9011 61 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, CDC37L1 NM O17913 -0.940979 100, 150, 175 or more genes or combinations of genes iden

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ing a functional miRNA to a cell, tissue, or subject. Modula TABLE 1-continued tion refers to the expression levels or activities of a gene or its related gene product (e.g., mRNA) or protein, e.g., the mRNA Genes with increased (positive values) or decreased (negative values) levels may be modulated or the translation of an mRNA may expression following transfection of human cancer cells with be modulated. Modulation may increase or up regulate a gene pre-miRhsa-miR-20a. or gene productor it may decrease or down regulate a gene or gene product (e.g., protein levels or activity). Gene Symbol RefSeq, Transcript ID (Pruitt et al., 2005) A log2 0028. Still a further embodiment includes methods of PTHLH NM 002820 NM 198964 -O.902774 administering an miRNA or mimic thereof, and/or treating a NM 198965 NM 198966 Subject or patient having, Suspected of having, or at risk of QKI NM OO6775 NM 206853 O.883687 developing a pathological condition comprising one or more NM 206854 NM 206855 of step (a) administering to a patient or Subject an amount of RAB22A NM O2O673 -126.569 RARRES1 NM 002888 NM 206963 0.715317 an isolated nucleic acid comprising a miR-20 nucleic acid RBKS NM 022128 -0.842482 sequence in an amount Sufficient to modulate expression of a RGC32 NM O14059 O.866694 cellular pathway; and (b) administering a second therapy, RHOC NM 175744 -0.874.504 wherein the modulation of the cellular pathway sensitizes the RNH1 NM OO2939 NM 203383 -10531 patient or subject, or increases the efficacy of a second NM 203384 NM 203385 therapy. An increase in efficacy can include a reduction in NM 203386 NM 203387 toxicity, a reduced dosage or duration of the second therapy, RRM2 NM 001034 -O-896356 or an additive or synergistic effect. A cellular pathway may S1 OOP NM 005.980 1.6654 SERF1A NM O21967 NM 022978 -0.777057 include, but is not limited to one or more pathway described SERF1B in Table 2 below or a pathway that is know to include one or SERPINE1 NM OOO602 -2.25784 more genes of Tables 1, 3, 4, and/or 5. The second therapy SESN1 NM O14454 O.845489 may be administered before, during, and/or after the isolated SGPL1 NM OO3901 -101.306 nucleic acid or miRNA is administered SKP2 NM OO5983 NM 032637 O.744696 0029. A second therapy can include administration of a SLC11A2 NM OOO617 O.845458 second miRNA ortherapeutic nucleic acid such as a siRNA or SLC1A4 NM OO3O38 O.721939 antisense oligonucleotide, or may include various standard SLC2A3 NM OO6931 O.879266 therapies, such as pharmaceuticals, chemotherapy, radiation SNAP23 NM 003825 NM 130798 O.791062 SPARC NM OO3118 1.391.99 therapy, drug therapy, immunotherapy, and the like. Embodi SPFH2 NM 001003790 NM 001003791 0.78.2553 ments of the invention may also include the determination or NM 007175 assessment of gene expression or gene expression profile for SPOCK NM 004598 -1.1917S the selection of an appropriate therapy. In a particular aspect, SQLE NM OO3129 O.773943 a second therapy is a chemotherapy. A chemotherapy can STC1 NM OO3155 -138313 include, but is not limited to paclitaxel, cisplatin, carboplatin, STX3A NM OO4177 O.809319 doxorubicin, oxaliplatin, larotaxel, taxol, lapatinib, doc SYNE1 NM O15293 NM 033071 -O.721107 etaxel, methotrexate, capecitabine, Vinorelbine, cyclophos NM 133650 NM 182961 phamide, gemcitabine, amrubicin, cytarabine, etoposide, TBC1D2 NM 018421 -O.96S6S TGFBR2 NM 001024847 NM OO3242 -O.924623 camptothecin, dexamethasone, dasatinib, tipifarnib, bevaci TP2 NM OO4817 NM 201629 19979 Zumab, sirolimus, temsirolimus, everolimus, lonafarnib, TM4SF2O NM O24795 O172 cetuximab, erlotinib, gefitinib, imatinib mesylate, rituximab, TM4SF4 NM 004617 -O.7OO123 trastuzumab, nocodazole, Sorafenib, Sunitinib, bortezomib, TM7SF1 NM OO3272 -1.8947 alemtuzumab, gemtuzumab, to situmomab or ibritumomab. TMEPAI NM O2O182 NM 199169 -102732 0030 Embodiments of the invention include methods of NM 19917O NM 199171 treating a Subject with a disease or condition comprising one TNFAIP6 NM 007115 -2.06788 TNFRSF10B NM OO3842 NM 147187 -O.725441 or more of the steps of (a) determining an expression profile TNRC9 XM O49037 O1681 of one or more genes selected from Table 1, 3, 4, and/or 5; (b) TSPAN8 NM 004616 0.858077 assessing the sensitivity of the Subject to therapy based on the TXLNA NM 175852 -0.7391.99 expression profile; (c) selecting a therapy based on the UEV3 NM 018314 -0.95S638 assessed sensitivity; and (d) treating the Subject using USP46 NM O22832 -1.54141 selected therapy. Typically, the disease or condition will have WANGL1 NM 138959 -O.809203 as a component, indicator, or result mis-regulation of one or VLDLR NM 001018056 NM 003383 -O.991.36 VTN NM 000638 298.43 more gene of Table 1, 3, 4, and/or 5. WNTSA NM OO3392 O6927 0031. In certain aspects, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ZBTB10 NM 023929 O.763786 miRNA may be used in sequence or in combination. For ZNF331 NM 018555 0.733817 instance, any combination of miR-20 with another miRNA ZNF395 NM 018660 O.710369 can be selected based on observing two given miRNAs share ZNF467 NM 207336 O.738748 a set of target genes or pathways listed in Tables 1, 2, 4 and 5 that are altered in a particular disease or condition. These two miRNAS may result in an improved therapy (e.g., reduced 0027. A further embodiment of the invention is directed to toxicity, greater efficacy, prolong remission, or other methods of modulating a cellular pathway comprising admin improvements in a Subjects condition), result in an increased istering to the cell an amount of an isolated nucleic acid efficacy, an additive efficacy, or a synergistic efficacy provid comprising a miR-20 nucleic acid sequence in an amount ing an additional or an improved therapeutic response. With Sufficient to modulate the expression, function, status, or state out being bound by any particular theory, synergy of two of a cellular pathway, in particular those pathways described miRNA can be a consequence of regulating the same genes or in Table 2 or the pathways known to include one or more related genes (related by a common pathway or biologic end genes from Table 1, 3, 4, and/or 5. Modulation of a cellular result) more effectively (e.g., due to distinct binding sites on pathway includes, but is not limited to modulating the expres the same target or related target(s)) and/or a consequence of sion of one or more gene(s). Modulation of a gene can include regulating different genes, but all of which have been impli inhibiting the function of an endogenous miRNA or provid cated in the same particular disease or condition. US 2009/0163434 A1 Jun. 25, 2009

0032. In certain aspects, miR-20 and let-7 can be admin 0039. In a further aspect, miR-20 and miR-143 are admin istered to patients with acute myeloid leukemia, breast istered to patients with astrocytoma, acute myeloid leukemia, carcinoma, bladder carcinoma, cervical carcinoma, colorec breast carcinoma, bladder carcinoma, cervical carcinoma, tal carcinoma, endometrial carcinoma, glioma, glioblastoma, colorectal carcinoma, endometrial carcinoma, glioma, glio gastric carcinoma, hepatocellular carcinoma, Hodgkin lym blastoma, gastric carcinoma, hepatocellular carcinoma, phoma, leukemia, melanoma, myxofibrosarcoma, multiple Hodgkin lymphoma, leukemia, melanoma, mantle cell lym myeloma, neuroblastoma, non-Hodgkin lymphoma, non phoma, multiple myeloma, non-Hodgkin lymphoma, non Small cell lung carcinoma, ovarian carcinoma, esophageal carcinoma, pancreatic carcinoma, prostate carcinoma, squa Small cell lung carcinoma, ovarian carcinoma, esophageal mous cell carcinoma of the head and neck, thyroid carcinoma, carcinoma, osteosarcoma, pancreatic carcinoma, prostate or urothelial carcinoma. carcinoma, squamous cell carcinoma of the head and neck, or 0033. Further aspects include administering miR-20 and thyroid carcinoma. miR-15 to patients with astrocytoma, acute myeloid leuke 0040. In still a further aspect, miR-20 and miR-147 are mia, breast carcinoma, bladder carcinoma, cervical carci administered to patients with astrocytoma, breast carcinoma, noma, colorectal carcinoma, endometrial carcinoma, glioma, bladder carcinoma, cervical carcinoma, colorectal carci glioblastoma, gastric carcinoma, hepatocellular carcinoma, noma, endometrial carcinoma, esophageal squamous cell car Hodgkin lymphoma, melanoma, mantle cell lymphoma, cinoma, glioma, glioblastoma, gastric carcinoma, hepatocel myxofibrosarcoma, multiple myeloma, neuroblastoma, non Hodgkin lymphoma, non-small cell lung carcinoma, ovarian lular carcinoma, Hodgkin lymphoma, leukemia, lipoma, carcinoma, esophageal carcinoma, osteosarcoma, pancreatic melanoma, mantle cell lymphoma, myxofibrosarcoma, mul carcinoma, prostate carcinoma, squamous cell carcinoma of tiple myeloma, non-Hodgkin lymphoma, non-Small cell lung the head and neck, or thyroid carcinoma. carcinoma, ovarian carcinoma, esophageal carcinoma, 0034. In still further aspects, miR-20 and miR-16 are osteosarcoma, pancreatic carcinoma, prostate carcinoma, administered to patients with astrocytoma, breast carcinoma, squamous cell carcinoma of the head and neck, or thyroid bladder carcinoma, colorectal carcinoma, endometrial carci carcinoma. noma, glioblastoma, gastric carcinoma, hepatocellular carci 0041. In yet another aspect, miR-20 and miR-188 are noma, Hodgkin lymphoma, melanoma, mantle cell lym administered to patients with astrocytoma, acute myeloid phoma, myxofibrosarcoma, multiple myeloma, non-small cell lung carcinoma, ovarian carcinoma, esophageal carci leukemia, breast carcinoma, bladder carcinoma, cervical car noma, pancreatic carcinoma, prostate carcinoma, squamous cinoma, colorectal carcinoma, endometrial carcinoma, cell carcinoma of the head and neck, or thyroid carcinoma. esophageal squamous cell carcinoma, glioma, glioblastoma, 0035 Aspects of the invention include methods where gastric carcinoma, hepatocellular carcinoma, leukemia, miR-20 and miR-21 are administered to patients with astro melanoma, multiple myeloma, non-Hodgkin lymphoma, cytoma, acute myeloid leukemia, breast carcinoma, bladder non-Small cell lung carcinoma, ovarian carcinoma, esoph carcinoma, colorectal carcinoma, endometrial carcinoma, ageal carcinoma, pancreatic carcinoma, prostate carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular car squamous cell carcinoma of the head and neck, or thyroid cinoma, melanoma, mantle cell lymphoma, neuroblastoma, carcinoma. non-Small cell lung carcinoma, ovarian carcinoma, esoph ageal carcinoma, pancreatic carcinoma, prostate carcinoma, 0042. In other aspects, miR-20 and miR-215 are adminis or squamous cell carcinoma of the head and neck. tered to patients with astrocytoma, acute myeloid leukemia, 0036. In still further aspects, miR-20 and miR-26a are breast carcinoma, bladder carcinoma, cervical carcinoma, administered to patients with acute myeloid leukemia, breast colorectal carcinoma, endometrial carcinoma, esophageal carcinoma, bladder carcinoma, cervical carcinoma, colorec Squamous cell carcinoma, glioma, glioblastoma, gastric car tal carcinoma, glioma, glioblastoma, gastric carcinoma, cinoma, hepatocellular carcinoma, Hodgkin lymphoma, leu hepatocellular carcinoma, leukemia, melanoma, multiple kemia, lipoma, melanoma, mantle cell lymphoma, myxofib myeloma, neuroblastoma, non-Hodgkin lymphoma, non rosarcoma, multiple myeloma, neuroblastoma, non-Hodgkin Small cell lung carcinoma, ovarian carcinoma, esophageal lymphoma, non-Small cell lung carcinoma, ovarian carci carcinoma, osteosarcoma, pancreatic carcinoma, or prostate noma, esophageal carcinoma, osteosarcoma, pancreatic car carcinoma. cinoma, prostate carcinoma, squamous cell carcinoma of the 0037. In yet further aspects, miR-20 and miR-34a are head and neck, thyroid carcinoma, or urothelial carcinoma. administered to patients with astrocytoma, acute myeloid 0043. In certain aspects, miR-20 and miR-216 are admin leukemia, breast carcinoma, bladder carcinoma, cervical car istered to patients with astrocytoma, breast carcinoma, cervi cinoma, colorectal carcinoma, endometrial carcinoma, cal carcinoma, colorectal carcinoma, endometrial carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular car glioma, glioblastoma, gastric carcinoma, hepatocellular car cinoma, Hodgkin lymphoma, leukemia, melanoma, mantle cinoma, Hodgkin lymphoma, leukemia, non-Hodgkin lym cell lymphoma, multiple myeloma, non-Hodgkin lymphoma, phoma, non-Small cell lung carcinoma, ovarian carcinoma, non-Small cell lung carcinoma, ovarian carcinoma, esoph ageal carcinoma, osteosarcoma, pancreatic carcinoma, pros esophageal carcinoma, osteosarcoma, prostate carcinoma, or tate carcinoma, squamous cell carcinoma of the head and squamous cell carcinoma of the head and neck. neck, thyroid carcinoma, or urothelial carcinoma. 0044. In a further aspect, miR-20 and miR-292-3p are 0038. In certain aspects, miR-20 and miR-126 are admin administered to patients with astrocytoma, acute myeloid istered to patients with astrocytoma, acute myeloid leukemia, leukemia, breast carcinoma, bladder carcinoma, cervical car breast carcinoma, bladder carcinoma, cervical carcinoma, cinoma, colorectal carcinoma, endometrial carcinoma, colorectal carcinoma, endometrial carcinoma, glioma, glio glioma, glioblastoma, gastric carcinoma, hepatocellular car blastoma, gastric carcinoma, hepatocellular carcinoma, cinoma, leukemia, lipoma, melanoma, myxofibrosarcoma, Hodgkin lymphoma, leukemia, melanoma, mantle cell lym multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, phoma, non-Hodgkin lymphoma, non-small cell lung carci non-Small cell lung carcinoma, ovarian carcinoma, esoph noma, ovarian carcinoma, esophageal carcinoma, osteosar ageal carcinoma, osteosarcoma, pancreatic carcinoma, pros coma, pancreatic carcinoma, prostate carcinoma, squamous tate carcinoma, squamous cell carcinoma of the head and cell carcinoma of the head and neck, or thyroid carcinoma. neck, thyroid carcinoma, or urothelial carcinoma. US 2009/0163434 A1 Jun. 25, 2009

0045. In still a further aspect, miR-20 and miR-331 are factor for a disease or condition) develop Such a condition. administered to patients with astrocytoma, acute myeloid Such a risk or propensity may indicate a treatment, increased leukemia, breast carcinoma, bladder carcinoma, cervical car monitoring, prophylactic measures, and the like. A nucleic cinoma, colorectal carcinoma, endometrial carcinoma, acid or probe set may comprise or identify a segment of a glioma, glioblastoma, gastric carcinoma, hepatocellular car corresponding mRNA and may include all or part of 1,2,3,4, cinoma, leukemia, melanoma, myxofibrosarcoma, multiple 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, myeloma, neuroblastoma, non-Hodgkin lymphoma, ovarian 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37,38, 39, carcinoma, esophageal carcinoma, osteosarcoma, pancreatic 40, 41,42, 43,44, 45,46,47, 48,49, 50, 51, 52,53,54, 55,56, carcinoma, prostate carcinoma, squamous cell carcinoma of 57, 58, 59, 60, 61, 62, 100, 200, 500, or more segments, the head and neck, or thyroid carcinoma. including any integer or range derivable there between, of a 0046. In yet a further aspect, miR-20 and miR-200b/c are gene or genetic marker, or a nucleic acid, mRNA or a probe administered to patients with breast carcinoma, cervical car representative thereofthat is listed in Tables 1, 3, 4, and/or 5 cinoma, colorectal carcinoma, glioma, glioblastoma, gastric or identified by the methods described herein. carcinoma, hepatocellular carcinoma, leukemia, lipoma, 0051 Certain embodiments of the invention are directed multiple myeloma, non-Small cell lung carcinoma, ovarian to compositions and methods for assessing, prognosing, or carcinoma, esophageal carcinoma, osteosarcoma, pancreatic treatingapathological condition in a patient comprising mea carcinoma, prostate carcinoma, squamous cell carcinoma of Suring or determining an expression profile of one or more the head and neck, or thyroid carcinoma. miRNA or marker(s) in a sample from the patient, wherein a 0047. It is contemplated that when miR-20 is given in difference in the expression profile in the sample from the combination with one or more other miRNA molecules, the patient and an expression profile of a normal sample or ref two different miRNAs may be given at the same time or erence expression profile is indicative of pathological condi sequentially. In some embodiments, therapy proceeds with tion and particularly cancer (e.g., In certain aspects of the one miRNA and that therapy is followed up with therapy with invention, the miRNAs, cellular pathway, gene, or genetic the other miRNA 1, 2, 3, 4, 5, 6,7,8,9, 10, 15, 20, 25, 30, 35, marker is or is representative of one or more pathway or 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, marker described in Table 1, 2, 3, 4, and/or 5, including any 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4, 5, 6, combination thereof. 7 days, 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 0052 Aspects of the invention include diagnosing, assess 12 months or any such combination later. ing, or treating a pathologic condition or preventing a patho 0048. Further embodiments include the identification and logic condition from manifesting. For example, the methods assessment of an expression profile indicative of miR-20 can be used to screen for a pathological condition; assess status in a cell or tissue comprising expression assessment of prognosis of a pathological condition; stage a pathological one or more gene from Table 1, 3, 4, and/or 5, or any combi condition; assess response of a pathological condition to nation thereof. therapy, or to modulate the expression of a gene, genes, or 0049. The term “miRNA is used according to its ordinary related pathway as a first therapy or to render a Subject sen and plain meaning and refers to a microRNA molecule found sitive or more responsive to a second therapy. In particular in eukaryotes that is involved in RNA-based gene regulation. aspects, assessing the pathological condition of the patient See, e.g., Carrington et al., 2003, which is hereby incorpo can be assessing prognosis of the patient. Prognosis may rated by reference. The term can be used to refer to the include, but is not limited to an estimation of the time or single-stranded RNA molecule processed from a precursor or expected time of Survival, assessment of response to a in certain instances the precursor itself or a mimetic thereof. therapy, and the like. In certain aspects, the altered expression 0050. In some embodiments, it may be useful to know of one or more gene or marker is prognostic for a patient whether a cell expresses a particular miRNA endogenously or having a pathologic condition, wherein the marker is one or whether such expression is affected under particular condi more of Table 1, 3, 4, and/or 5, including any combination tions or when it is in a particular disease state. Thus, in some thereof. embodiments of the invention, methods include assaying a cell or a sample containing a cell for the presence of one or TABLE 2 more miRNA marker gene or mRNA or other analyte indica tive of the expression level of a gene of interest. Conse Significantly affected functional cellular pathways following quently, in some embodiments, methods include a step of hsa-miR-20a over-expression in human cancer cells. generating an RNA profile for a sample. The term “RNA Gene profile' or “gene expression profile' refers to a set of data Number Pathway Functions regarding the expression pattern for one or more gene or 17 Cellular Movement, Cellular Growth and Proliferation, genetic marker in the sample (e.g., a plurality of nucleic acid Cardiovascular System Development and Function probes that identify one or more markers or genes from Tables 14 Cell Morphology, Cardiovascular System Development and 1, 3, 4, and/or 5); it is contemplated that the nucleic acid Function, Cell-To-Cell Signaling and Interaction profile can be obtained using a set of RNAs, using for 13 Endocrine System Disorders, Small Molecule Biochemistry, Immune Response example nucleic acid amplification or hybridization tech 13 Cardiovascular System Development and Function, Tissue niques well know to one of ordinary skill in the art. The Morphology, Genetic Disorder difference in the expression profile in the sample from a 12 Lipid , Molecular Transport, Small Molecule patient and a reference expression profile, Such as an expres Biochemistry 9 Developmental Disorder, Tumor Morphology, Cancer sion profile from a normal or non-pathologic sample, or a 1 Cell Signaling, Molecular Transport, Neurological Disease digitized reference, is indicative of a pathologic, disease, or 1 Cancer, Cell Cycle, Skeletal and Muscular Disorders cancerous condition. In certain aspects the expression profile is an indicator of a propensity to or probability of (i.e., risk US 2009/0163434 A1 Jun. 25, 2009 10

TABLE 3 Predicted target genes of hsa-miR-20a. RefSeq, Transcript ID (Pruitt et al., 2005) Description NM O14444 Gamma tubulin ring complex protein (76p gene) NM 130786 alpha 1 B-glycoprotein NM 144670 alpha-2-macroglobulin-like 1 NM 001086 arylacetamide deacetylase NM 020792 arylacetamide deacetylase-like 1 NM 016228 alpha-aminoadipate aminotransferase NM 020745 Alanyl-tRNA synthetase like NM OOO663 4-aminobutyrate aminotransferase precursor NM OO55O2 ATP-binding cassette, Sub-family A member 1 NM 080282 ATP-binding cassette, Sub-family A, member 10 NM 019624 ATP-binding cassette, Sub-family B (MDR/TAP), NM 172024 ATP-binding cassette protein C13 isoform b NM 005688 ATP-binding cassette, Sub-family C, member 5 NM OO5164 ATP-binding cassette, Sub-family D, member 2 NM 002940 ATP-binding cassette, Sub-family E, member 1 NM 004.827 ATP-binding cassette, Sub-family G, member 2 NM 0221 69 ATP-binding cassette, subfamily G, member 4 NM 031295 Abhydrolase domain containing 11 isoform 4 NM 032859 Hypothetical protein LOC84945 NM 007011 alphabeta hydrolase domain containing protein NM O22060 Abhydrolase domain containing 4 BI1 NM 001012750 abil-interactor 1 isoform b BL NM OO5157 V-abl Abelson murine leukemia viral oncogene BLIM1 NM OO10O3407 -binding LIM protein 1 isoform b BR NM 001092 Active breakpoint cluster region-related BT NM 013375 activator of basal transcription 1 BTB1 NMO32548 Ankyrin repeat and BTB (POZ) domain containing 1 CAD8 NM 014384 acyl-Coenzyme A dehydrogenase family, member 8 CADSB NM OO1609 acyl-Coenzyme A dehydrogenase, short branched ACIN1 NM O14977 apoptotic condensation inducer 1 ACPL2 NM 152282 acid phosphatase-like 2 ACPP NM 001099 prostatic acid phosphatase precursor ACSL1 NM OO1995 acyl-CoA synthetase long-chain family member 1 ACSL4 NM 004.458 acyl-CoA synthetase long-chain family member 4 ACSM1 NM 052956 acyl-CoA synthetase medium-chain family member ACTR2 NM 001005386 actin-related protein 2 isoform a ACVR1B NM 004302 activin A type IB receptor isoform a precursor DAM19 NM 033274 DAM metallopeptidase domain 19 isoform 2 DAM21 NM OO3813 DAM metallopeptidase domain 21 preproprotein DAM33 NM O25220 DAM metallopeptidase domain 33 isoform alpha DAM9 NM 001005845 DAM metallopeptidase domain 9 isoform 2 DAMTS3 NM 014243 DAM metallopeptidase with thrombospondin type 1 DAMTSS NM 007038 DAM metallopeptidase with thrombospondin type 1 DAMTSL2 NM 014694 DAMTS-like 2 DAMTSLS NM 213604 rombospondin, type I, domain containing 6 DAR NM 001025107 enosine deaminase, RNA-specific isoform d DARB1 NM OO1033049 RNA-specific adenosine deaminase B1 isoform 4 DAT1 NM 012091 8. enosine deaminase, tRNA-specific 1 DCY1 NM 021116 brain adenylate cyclase 1 DCY6 NM O15270 enylate cyclase 6 isoform a DCY9 NM 001116 enylate cyclase 9 DD1 NM 001119 dducin 1 (alpha) isoform a HFE1 NM 144650 cohol dehydrogenase, iron containing, 1 POR2 NM O24551 iponectin receptor 2 M 2 NM 024.866 renomedulin 2 precusor ORA2B NM OOO676 enosine A2b receptor PN NM O25225 diponutrin PRHL2 NM 017825 DP-ribosylhydrolase like 2 RA1B NM OOO679 pha-1B-adrenergic receptor NM OOO681 pha-2A-adrenergic receptor NM OOO682 pha-2B-adrenergic receptor NM OOOO25 adrenergic, beta-3-, receptor NM OOOO26 adenylosuccinate lyase NM 1532O7 AE binding protein 2 AR 3 NM 201252 aflatoxin B1 aldehyde reductase 3 NM 005935 Myeloid lymphoid or mixed-lineage leukemia NM 002025 Fragile X mental retardation 2 NM O14423 ALL1 fused gene from 5q31 GA NM 000027 aspartylglucosaminidase precursor A.G B L 2 NM O24783 ATP/GTP binding protein-like 2 US 2009/0163434 A1 Jun. 25, 2009 11

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq Gene Transcript ID Symbol (Pruitt et al., 2005) Description AGGF1 NM 018046 angiogenic factor VG5C AGPAT4 NM 001012733 1-acylglycerol-3-phosphate O-acyltransferase 4 AGPATS NM 018361 1-acylglycerol-3-phosphate O-acyltransferase 5 AGTPBP1 NM O15239 ATP/GTP binding protein 1 AGTR2 NM OOO686 angiotensin II receptor, type 2 AGXT2L1 NM 031279 alanine-glyoxylate aminotransferase 2-like 1 AHCTF1 NM 015446 Transcription factor ELYS AHCY NM OOO687 S-adenosylhomocysteine hydrolase AHI1 NM O17651 Jouberin AHNAK NM OO1620 AHNAKnucleoprotein isoform 1 AICDA NM 020661 activation-induced cytidine deaminase AIM1 NM OO1624 absent in melanoma 1 AIPL1 NM OO 1033054 aryl hydrocarbon receptor interacting AJAP1 NM 018836 transmembrane protein SHREW1 AK1 NM 000476 adenylate kinase 1 AKS NM 012093 adenylate kinase 5 isoform 2 AKAP11 NM O16248 A-kinase anchor protein 11 isoform 1 AKAP13 NM OO6738 A-kinase anchor protein 13 isoform 1 AKAP6 NM OO4274 A-kinase anchor protein 6 AKAP9 NM O05751 A-kinase anchor protein 9 isoform 2 AKR1D1 NM OO5989 aldo-keto reductase family 1, member D1 AKR7A2 NM OO3689 aldo-keto reductase family 7, member A2 AKT3 NM OO5465 V-akt murine thymoma viral oncogene homolog 3 ALDH1A3 NM OOO693 aldehyde dehydrogenase 1A3 ALDH3A2 NM OOO382 aldehyde dehydrogenase 3A2 isoform 2 ALDH3B1 NM 000694 aldehyde dehydrogenase 3B1 isoform a ALDH8A1 NMO22568 aldehyde dehydrogenase 8A1 isoform 1 ALDH9A1 NM OOO696 aldehyde dehydrogenase 9A1 ALDOC NM OO5165 fructose-bisphosphate aldolase C ALKBH4 NM O17621 Hypothetical protein LOC54784 ALKBHS NM O17758 Hypothetical protein LOC54890 ALOX1SB NM 001141 arachidonate 15-lipoxygenase, second type ALPK1 NM O25144 alpha-kinase 1 ALPP NM OO1632 placental alkaline phosphatase precursor ALS2CL NM 147129 ALS2 C-terminal like isoform 1 ALS2CR13 NM 173511 Amyotrophic lateral sclerosis 2 (juvenile) ALS2CR1S NM 138468 Icaé9-related protein ALS2CR19 NM 0571 77 Amyotrophic lateral sclerosis 2 (juvenile) ALX4 NM O21926 aristaless-like homeobox 4 AMELX NM 001142 amelogenin (X ) isoform 1 precursor AMELY NM 001143 amelogenin (Y chromosome) precursor AMID NM 032797 apoptosis-inducing factor (AIF)-like AMIGO2 NM 181847 amphoterin induced gene 2 AMMECR1 NM 001025580 AMMECR1 protein isoform 2 AMOTL1 NM 130847 angiomotin like 1 AMPD2 NM 004037 adenosine monophosphate deaminase 2 (isoform L) AMPD3 NM 000480 erythrocyte adenosine monophosphate deaminase AMZ1. NM 133463 archaemetZincin-1 ANAPC11 NM 0010O2244 APC11 anaphase promoting complex subunit 11 ANGEL.1 NM O15305 angelhomolog 1 ANGEL2 NM 144567 LOC90806 protein ANGPTL7 NM 021146 Angiopoietin-like 7 ANK2 NM 001148 Ankyrin 2 isoform 1 ANKFY1 NM 016376 Ankyrin repeat and FYVE domain containing 1 ANKH NM 054027 ankylosis, progressive homolog ANKK NM 178510 Ankyrin repeat and kinase domain containing 1 ANKRA2 NM O23O39 Ankyrin repeat, family A (RFXANK-like), 2 ANKRD10 NM O17664 Ankyrin repeat domain 10 ANKRD11 NM 013275 Ankyrin repeat domain 11 ANKRD12 NM O15208 Ankyrin repeat domain 12 ANKRD13C NM O3O816 Ankyrin repeat domain 13C ANKRD15 NM O15158 Ankyrin repeat domain protein 15 isoform a ANKRD16 NM O19046 Ankyrin repeat domain 16 isoform a ANKRD25 NM O15493 Ankyrin repeat domain 25 ANKRD28 NM O15199 Ankyrin repeat domain 28 ANKRD29 NM 173505 Ankyrin repeat domain 29 ANKRD38 NM 181712 Ankyrin repeat domain 38 ANKRD42 NM 182603 Ankyrin repeat domain 42 ANKRD44 NM 153697 Hypothetical protein DKFZp434D2328 ANKRDSO NM 020337 Hypothetical protein LOC57182 US 2009/0163434 A1 Jun. 25, 2009 12

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description ANKRD9 NM 152326 Ankyrin repeat domain 9 ANKS1A NM O15245 Ankyrin repeat and sterile alpha motif domain ANKS1B NM 020140 Cajalin 2 isoform c ANKS4B NM 145865 harmonin-interacting ankyrin-repeat containing ANTXR1 NM 018153 Tumor endothelial marker 8 isoform 3 precursor ANUBL1 NM 174890 AN1, ubiquitin-like, homolog ANXA13 NM OO10O3954 Annexin A13 isoform b ANXA7 NM OO1156 Annexin VII isoform 1 NM 153042 Amine oxidase (flavin containing) domain 1 NM OO1030007 Adaptor-related protein complex 1, gamma 1 NM OO3916 Adaptor-related protein complex 1 sigma 2 NM OO1030006 Adaptor-related protein complex 2, beta 1 NM OO3938 Adaptor-related protein complex 3, delta 1 NM 007077 Adaptor-related protein complex 4, sigma 1 NM 173075 Amyloid beta A4 precursor protein-binding, NM 006051 Amyloid beta precursor protein-binding, family NM OOOO38 adenomatosis polyposis coli NM 153000 adenomatosis polyposis coli down-regulated 1 NM 001641 APEX nuclease NM OO6595 apoptosis inhibitor 5 NM 145699 phorbolin 1 NM 001006666 apolipoprotein B mRNA editing enzyme, catalytic NM 203454 apolipoprotein B mRNA editing enzyme, catalytic NM OO3661 apolipoprotein L1 isoform a precursor NM O3O817 Hypothetical protein LOC81575 NM 000484 Amyloid beta A4 protein precursor, isoform a NMOO6380 Amyloid beta precursor protein-binding protein NM 012096 Adaptor protein containing pH domain, PTB domain NM 133456 Apical protein 2 NM OO1650 aquaporin 4 isoform a NM 020980 aquaporin 9 NM OO1655 chain NM 012402 DP-ribosylation factor interacting protein 2 NM 00101.2659 A.ypothetical protein LOC503582 HGAP1 NM 004308 ho GTPase activating protein 1 HGAP12 NM 018287 ho GTPase activating protein 12 HGAP18. NM 033515 ho GTPase activating protein 18 HGAP24 NM 031305 ho GTPase activating protein 24 HGAP26 NM O15071 TPase regulator associated with the focal HGAPS NM OO 1030055 ho GTPase activating protein 5 isoform a HGAP6 NM OO6125 ho GTPase activating protein 6 isoform 3 HGEF10 NM 014629 hoguanine nucleotide exchange factor 10 HGEF11 NM 014784 hoguanine nucleotide exchange factor (GEF) 11 HGEF18 NM O15318 ho-specific guanine nucleotide exchange factor HGEF3 NM O19555 hoguanine nucleotide exchange factor 3 HGEF6 NM 004840 Ra cCdc42 guanine nucleotide exchange factor 6 HGEF7 NM OO3899 Rho guanine nucleotide exchange factor 7 isoform D4A NM 002892 retinoblastoma-binding protein 1 isoform I NM 016374 AT rich interactive domain 4B isoform 1 NM 001177 ADP-ribosylation factor-like 1 NM 173664 ADP-ribosylation factor-like 10 NM 144996 ADP-ribosylation factor-like 2-like 1 isoform 2 NM OO5738 A. DP-ribosylation factor-like 4A NM 005.737 A. DP-ribosylation factor-like 4C NM 014154 armadillo repeat containing 8 isoform 1 NT 2 NM O14862 aryl hydrocarbon receptor nuclear translocator NM OO6628 Cyclic AMP phosphoprotein, 19 Kd NM 001025068 Cyclic AMP-regulated phosphoprotein, 21 kD NM 152285 Arrestin domain containing 1 NM 000046 Arylsulfatase B isoform 1 precursor s R NM OO1669 Arylsulfatase D isoform a precursor NM O24590 Arylsulfatase J S 1 NM 016442 type 1 tumor necrosis factor receptor shedding NM 004315 N-acylsphingosine amidohydrolase (acid NM 001010887 N-acylsphingosine amidohydrolase 3-like NM O14435 N-acylsphingosine amidohydrolase-like protein NM O16114 Ankyrin repeat and SOCS box-containing protein NM O24701 Ankyrin repeat and SOCS box-containing protein NM 080874 Ankyrin repeat and SOCS box-containing protein NM O17873 Ankyrin repeat and SOCS box-containing 6 isoform US 2009/0163434 A1 Jun. 25, 2009 13

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description ASB7 NM 198243 Ankyrin repeat and SOCS box-containing protein 7 ASB9 NM OO 1031739 Ankyrin repeat and SOCS box-containing 9 isoform ASCIZ NM O15251 ATM ATR-Substrate Chk2-Interacting Zn2+-finger ASF1A NM 014034 ASF1 anti-silencing function 1 homolog A ASL NM 000048 argininosuccinate lyase isoform 1 ASTN NM 004319 astrotactin isoform 1 ATAD2 NM 014109 two AAA domain containing protein ATF5 NM 012068 activating transcription factor 5 ATF7IP2 NM O24997 activating transcription factor 7 interacting ATG10 NM 031482 APG10 autophagy 10-like ATG12 NM 004707 APG12 autophagy 12-like ATG16L1 NM O17974 APG 16 autophagy 1.6-like isoform 2 ATG4B NM 013325 APG4 autophagy 4 homolog B isoform a ATGS NM 004849 APG5 autophagy 5-like ATM NM 000051 Ataxiatelangiectasia mutated protein isoform 1 ATOH8 NM 032827 Atonal homolog 8 ATP11A NM O15205 ATPase, Class VI, type 11A isoform a ATP12A NM OO1676 ATPase, H+/K+ transporting, nongastric, alpha ATP1A2 NM 000702 Na+K+-ATPase alpha 2 subunit proprotein ATP2B1 NM OO1 OO1323 plasma membrane calcium ATPase 1 isoform 1 a ATP2B2 NM OO1OO1331 plasma membrane calcium ATPase 2 isoform a ATP6VOE NM OO3945 A TPase, H-- transporting, lysosomal, VO subunit ATP6V1D NM O15994 H(+)-transporting two-sector ATPase ATP7B NM 000053 ATPase, Cu++ transporting, beta polypeptide ATP8B4 NM O24837 ATPase class I type 8B member 4 ATP9A NM OO6045 ATPase, Class II, type 9A ATPAF1 NM O22745 ATP synthase mitochondrial F1 complex assembly ATPBD1B NM 018066 ATP binding domain 1 family, member B ATPBD1C NM 016301 ATP binding domain 1 family, member C ATRNL1 NM 207303 attractin-like 1 ATXN1 NM OOO332 Ataxin 1 ATXN3 NM 001024631 Ataxin 3 isoform 3 B2M NM 004.048 beta-2-microglobulin precursor B3GALNT2 NM 152490 UDP-GalNAc:betaGlcNAc beta B3GALT2 NM 003783 UDP-Gal:betaGlcNAc beta B3GALTS NM OO6057 UDP-Gal:betaGlcNAc beta B3GNTS NM 032047 beta-1,3-N-acetylglucosaminyltransferase bOnT-5 B3Gn-T6 NM 1387O6 beta-1,3-N-acetylglucosaminyltransferase B4GALT2 NM 001005417 DP-Gal:betaGlcNAc beta 14 B4GALTS NM OO4776 DP-Gal:betaGlcNAc beta 14 B4GALT6 NM 004775 DP-Gal:betaGlcNAc beta 14 BAALC NM 001024372 brain and acute leukemia, cytoplasmic isoform 2 BACH2 NM 021813 BTB and CNC homology 1, basic leucine zipper BAG1 NM 004323 BCL2-associated athanogene isoform 1L. BAGS NM 00101504.8 BCL2-associated athanogene 5 isoform b BAGE NM 001187 B melanoma antigen BAGE4 NM 181704 B melanoma antigen family, member 4 BAHD1 NM O14952 Bromo adjacent homology domain containing 1 BAMBI NM 012342 BMP and activin membrane-bound inhibitor BAPX1 NM 001189 Bagpipe homeobox 1 BCAP29 NM OO10O8405 B-cell receptor-associated protein BAP29 isoform BCAS1 NM OO3657 Breast carcinoma amplified sequence 1 BCAS2 NM OO5872 Breast carcinoma amplified sequence 2 BCL11B NM 022898 B-cell CLL/lymphoma 11B isoform 2 BCL2 NM 000633 B-cell lymphoma protein 2 alpha isoform BCL2L11 NM OO6538 BCL2-like 11 isoform 6 BCL2L2 NM 004.050 BCL2-like 2 protein BCL6 NM OO1706 B-cell lymphoma 6 protein BCL6B NM 181844 B-cell CLL/lymphoma 6, member B ( finger BDH2 NM O2O139 3-hydroxybutyrate dehydrogenase, type 2 BET1 NM OO5868 Blocked early in transport 1 BET1L. NM O16526 Blocked early in transport 1 homolog (S. BFAR NM O16561 apoptosis regulator BHLHB3 NM 030762 basic helix-loop-helix domain containing, class BHMT2 NM O17614 betaine-homocysteine methyltransferase 2 BICD2 NM OO10O3800 bicaudal Dhomolog 2 isoform 1 BIRC1 NM OO4536 baculoviral IAP repeat-containing 1 BIRC4 NM 001167 baculoviral IAP repeat-containing protein 4 BIRC4BP NM 017523 XIAP associated factor-1 isoform 1 BIRCS NM 00101.2270 baculoviral IAP repeat-containing protein 5 US 2009/0163434 A1 Jun. 25, 2009 14

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description BLZF1 NM OO3666 basic leucine Zipper nuclear factor 1 BMP8B NM OO1720 bone morphogenetic protein 8B preproprotein BMPR2 NM 001204 bone morphogenetic protein receptor type II BMX NM OO1721 BMX non-receptor tyrosine kinase BNC2 NM O17637 basonuclin 2 BNIP2 NM 004330 BCL2/adenovirus E1B 19 kD interacting protein 2 BNIP3L NM 004331 BCL2/adenovirus E1B 19 kD-interacting protein BNIPL NM 138279 BCL2/adenovirus E1B 19 kD interacting protein BPGM NM OO1724 2,3-bisphosphoglycerate mutase BPHL NM 004332 biphenyl hydrolase-like BPNT 1 NM OO6085 3'(2),5'-bisphosphate nucleotidase 1 BRCA1 NM OO7294 Breast cancer 1, early onset isoform 1 BRCA2 NM 000059 Breast cancer 2, early onset BRD1 NM 014577 bromodomain containing protein 1 BRMS1L, NM 032352 Breast cancer metastasis-Suppressor 1-like BRWD1 NM OO1007246 bromodomain and WD repeat domain containing 1 BSCL2 NM 032667 Seipin BSDC1 NM 018045 BSD domain containing 1 BTBD10 NM 032320 K+ channel tetramerization protein BTBD15 NM 014155 BTB (POZ) domain containing 15 BTBD7 NM OO10O2860 BTB (POZ) domain containing 7 isoform 1 NM OO1731 B-cell translocation protein 1 NM OO6806 B-cell translocation gene 3 NM OO1732 Butyrophilin, subfamily 1, member A1 NM 007048 Butyrophilin, subfamily 3, member A1 NM 007047 Butyrophilin, subfamily 3, member A2 precursor BUB1 NMOO4336 BUB1 budding uninhibited by benzimidazoles 1 VES NM 007073 Blood vessel epicardial substance Oor O4 NM 173473 Hypothetical protein LOC119504 Oor 14 NM 00101.0911 Hypothetical protein LOC399726 Oor 18 NM 018017 CTCL tumor antigen L14-2 Oor 29 NM 207321 Hypothetical protein LOC142827 Oor 37 NM O15608 erythroid differentiation-related factor 1 Oor 22 NM 032804 bothetical protein LOC84890 Oor A2 NM 138357 bothetical protein LOC90550 Oor A6 NM 153810 pothetical protein LOC143384 Oor S4 NM 022153 pothetical protein LOC64115 Oor 57 NM O251.25 bothetical protein LOC8O195 Oor 58 NM 032333 pothetical protein LOC84293 Oor 72 NM 144984 pothetical protein LOC196740 isoform 2 Oor 76 NM 024541 bothetical protein LOC79591 Oor 78 NM OO1002759 pothetical protein LOC119392 isoform a Oor NM 001012711 pothetical protein LOC404216 Oor 96 NM 198515 bothetical protein LOC374355 Oor 97 NM 024948 romosome 10 open reading frame 97 1or NM O22761 bothetical protein LOC64776 1or 30 NM O2O193 SY protein 1or 38 NM 212555 bothetical protein LOC399967 1or A9 NM 001003678 pothetical protein LOC79096 isoform 4 1or S4 NM 014039 bothetical protein LOC28970 1or 55 NM 207428 bothetical protein LOC399879 1or 63 NM 199124 pothetical protein LOC79864 isoform 2 1or 69 NM 152314 pothetical protein LOC120196 2Or 31 NM 032338 pothetical protein LOC84298 2Or 36 NM 182558 pothetical protein LOC283422 2Or NM 021934 bothetical protein LOC60673 2Or NM O24738 bothetical protein LOC79794 2Or NM 153685 bothetical protein LOC1965.00 3 or NM 020456 bothetical protein LOC57213 4or O1 NM O17799 bothetical protein LOC54916 4or O3 NM 018036 bothetical protein LOC55102 4or 05 NM 018168 bothetical protein LOC55195 4or O8 NM 018229 bothetical protein LOC55745 4or 11 NM O15962 bothetical protein LOCS1077 4or 19 NM O17924 romosome 14 open reading frame 119 4or 26 NM 080664 pothetical protein LOC112487 4or 29 NM 0164.72 bothetical protein LOC51527 4or 33 NM O22067 bothetical protein LOC63894 4or 38 NM O24558 bothetical protein LOC79.609 4or 43 NM 145231 pothetical protein LOC90141 US 2009/0163434 A1 Jun. 25, 2009 15

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description 4orf145 NM 1524.46 Chromosome 14 open reading frame 145 4orf150 Hypothetical protein LOC112840 4orf153 pothetical protein LOC84334 4orf24 O hetical protein LOC28363S 4orf28 s pothetical protein LOC122S25 4orf32 MA. P K-interacting and spindle-stabilizing 4orf243 pothetical protein LOC91748 4orf244 O hetical protein LOC145483 Sorf17 s pothetical protein LOCS7184 Sorf2O DNA helicase homolog PIF1 Sorf32 pothetical protein LOC145858 Sorf240 pothetical protein LOC1232O7 Sorf241 pothetical protein LOC84529 6orf28 pothetical protein LOC65259 6orf34 romosome 16 open reading frame 34 6orf245 pothetical protein LOC89927 6orf54 NM 175900 pothetical protein LOC283897 6orf58 NM 022744 pothetical protein LOC647SS 6orf59 NM O251.08 pothetical protein LOC8O178 7orf27 NM 020914 Chromosome 17 open reading frame 27 7orf37 NM 032339 Chromosome 17 open reading frame 37 7orf59 NM 024052 tlypone ical protein LOC79018 7orf2O NM 018428 hepatocellular carcinoma-associated antigen 66 7orf53 NM 024032 OleC8 Ocil LOC78995 7orf62 NM OO 1033046 pothetical protein LOC7941S 7orf69 NM 152466 pothetical protein LOC147081 7orf73 NM O17928 O hetical protein LOCSSO18 7orf77 NM 152460 pothetical protein LOC146723 8or NM 001003674 pothetical protein LOC753 isoform gamma 1 8orf16 NM 153010 pothetical protein LOC147429 8orf17 NM 153211 O hetical protein LOC125488 8orf19 NM 152352 s pothetical protein LOC125228 8orf25 NM OO10O8239 romosome 18 open reading frame 25 isoform b 8orf26 NM 173629 pothetical protein LOC284254 8orf245 NM 032933 O hetical protein LOC85O19 9orf12 NM 031448 s pothetical protein LOC83636 isoform 2 9orf2 NM 003796 B5-mediating protein isoform a 9orf20 NM 033513 9. r e trap ROSAb-geo 22 9orf31 NM 00101.4373 pothetical protein LOC4O4664 GALT1 NM O2O156 e synthase, orf107 NM 014388 pothetical protein LOC27042 orf108 NM O24595 O hetical protein LOC79647 orf110 NM 1785.50 pothetical protein LOC339S12 orf116 NM O23938 cifically- androgen-regulated protein orf130 NM 00101.0980 pothetical protein LOC4OO746 orf13S NM 024037 pothetical protein LOC79OOO orf138 NM 001025493 O hetical protein LOCS744O6 orf150 NM 145278 pothetical protein LOC148823 orf151 NM OO1032363 C romosome 1 open reading frame 151 protein orf15S NM 033319 pothetical protein LOC91687 orf171 NM 138467 pothetical protein LOC127253 orf173 NM OO10O2912 pothetical protein LOC127254 orf176 NM O22774 pothetical protein LOC64789 orf178 NM 001010922 poptotic Bcl-2 p rotein isoform a orf183 NM O19099 Hypothetical protein LOC55924 isoform 1 orf19 NM 052965 Hypothetical protein LOC116461 orf21 NM 030806 Chromosome 1 open reading frame 21 orf24 NM O22083 niban protein isoform 1 orf26 NM O17673 hetical protein LOC54823 orf32 NM 1993.51 hetical protein LOC387597 orf33 NM 016183 omal protein P0-like protein orf242 NM O19060 chromosome 1 open reading frame 42 orf63 NM 020317 hypo hetical protein LOC57035 isoform 2 orf69 NM 001010867 hypo hetical protein LOC200205 orf76 NM 173509 hypo hetical protein MGC16664 orf&3 NM 153035 hypo hetical protein LOC127428 orf&4 NM 1825.18 RP11-506B15.1 protein isoform 3 orf NM O14283 chromosome 1 open reading frame 9 protein Orf6 NM 145257 hetical protein LOC126731 QDC1 NM OO10O2259 omain containing 1 isoform 1 US 2009/0163434 A1 Jun. 25, 2009 16

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description NM 03.1911 C1q and tumor necrosis factor related protein 7 NM 012261 chromosome 20 open reading frame 103 precursor NM 080821 hypotnetical protein LOC116151 NM 080616 hypothetical protein LOC14O688 NM 080627 hypothetical protein LOC140710 isoform 1 NM 018152 hypothetical protein LOCSS184 NM O24331 hypothetical protein LOC791.83 NM OO1033086 hypothetical protein LOC140733 isoform 1 NM 033421 Sorting nexin 21 isoform a NM 024918 hypothetical protein LOC7998O NM 080829 hypothetical protein LOC140876 NM 022106 hypothetical protein LOC63939 NM 018347 hypothetical protein LOC55317 NM 016407 hypothetical protein LOC51507 NM O22099 hypothetical protein LOC63930 NM 199050 hypothetical protein LOC25966 NM 001006116 hypothetical protein LOCS4O67 NM O17833 hypothetical protein LOCS4943 NM 05818O hypothetical protein LOC54058 isoform 1 NM 019596 hypothetical protein LOCS6245 NM 0581.87 chromosome 21 open reading frame 63 NM 145328 GC-rich sequence DNA-binding factor candidate NM 018277 hypothetical protein LOCSS264 NM 00100988O hypothetical protein LOC23313 isoform b NM 173545 hypothetical protein LOC2OOSS8 NM 144706 hypothetical protein LOC150590 NMO24293 hypothetical protein LOC791.37 NM 001024676 open reading frame 19 NM O23016 hypothetical protein LOC65124 NM 016085 apoptosis related pro ein 3 isoform a NM O03203 hypothetical protein LOC6936 NM O16589 hypothetical protein LOCS1300 NM 152531 hypothetical protein LOC1520O2 NM OO7354 putative GR6 protein NM 032898 hypothetical protein LOC84984 NM 178342 AP20 region protein isoform E NM 173824 hypone ical protein LOC285237 NM O24616 TPA-induced transmembrane protein NM OO1.007534 hypothetical protein LOC285311 NM 198562 hypothetical protein LOC37S341 NM O15224 retinoblastoma-assoc iated protein 140 NM 173654 AER61 glycosyltransferase NM O2O231 hypothetical protein LOCS6983 NM 205857 FBI4 protein NM 001029998 hypothetical protein LOC84068 isoform b NM O24511 hypothetical protein LOC79441 NM OO1735 complement compon ent 5 NM 018356 hypothetical protein LOC55322 NM 001029863 hypothetical protein LOC387263 NM 145316 hypothetical protein LOC221468 NM 024909 hypothetical protein LOC79969 isoform 2 NM 018132 hypothetical protein LOCSS166 NM O14070 STG protein NM 152551 NM 206834 hypothetical protein LOC404220 NM O250O2 hypothetical protein LOC8OO69 NM 018452 hypothetical protein LOCSS836 NM 013397 over-expressed breas tumor protein NM 024929 hypothetical protein LOC79992 NM 173562 hypothetical protein LOC222658 NM 203395 open reading frame 71 NM O21945 ion transporter protei l NM O17909 hypothetical protein LOC55005 NM O25059 hypothetical protein LOC8O129 NM OOO587 complement compon ent 7 precursor NM 032831 hypothetical protein LOC80228 NM 138434 hypothetical protein LOC113763 NM OOO562 complement compon ent 8, alpha polypeptide NM 004337 hypothetical protein LOC734 NM 016458 brain protein 16 US 2009/0163434 A1 Jun. 25, 2009 17

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Transcript ID (Pruitt et al., 2005) Description NM 177965 hypothetical protein LOC157657 NM 152416 hypothetical protein LOC137682 NM 019607 hypothetical protein LOC56260 NM 173518 hypothetical protein LOC157777 NM OO1031839 hypothetical protein LOC606553 NM OO1031728 hypothetical protein LOC84904 isoform 1 NM 020207 stretch responsive protein 278 isoform a NM 178448 hypothetical protein LOC89958 NM O17998 hypothetical protein LOC55071 NM 032012 hypothetical protein LOC23731 NM 032307 hypothetical protein LOC84267 NM 152569 hypothetical protein LOC157983 NM 145005 hypothetical protein LOC203228 isoform b NM 001025780 chromosome 9 open reading frame 77 isoform 2 NM 016482 chromosome 9 open reading frame 78 isoform 1 NM 021218 hypothetical protein LOC58493 NM O24828 hypothetical protein LOC79886 NM 182505 hypothetical protein LOC138241 isoform a NM 022833 hypothetical protein LOC64855 NM O2O178 carbonic anhydrase X NM 004.056 carbonic anhydrase VIII NM 138375 CdkS and Ablenzyme substrate 1 NM 016366 calcium binding protein 2 isoform 1 NM O14405 voltage-dependent calcium channel gamma-4 NM OO5831 calcium binding and coiled-coil domain 2 NM 004342 caldesmon 1 isoform 2 NMOO1017440 calneuron 1 CAMK1D NM O2O397 calcium calmodulin-dependent protein kinase ID CAMK2D NM 172127 calcium calmodulin-dependent protein kinase II CAMK2G NM OO1222 calcium calmodulin-dependent protein kinase II CAMK2N1 NM 018584 calcium calmodulin-dependent protein kinase II CAMK2N2 NM 033259 CaM-KII inhibitory protein CAMKK1 NM 032294 calcium calmodulin-dependent protein kinase 1 CAMSAP1 NM 015447 calmodulin regulated spectrin-associated protein CAMSAP1L1 NM 203459 calmodulin regulated spectrin-associated protein CAMTA1 NM O15215 calmodulin-binding transcription activator 1 CAMTA2 NM O15099 calmodulin binding transcription activator 2 CANX NM 001024649 calnexin precursor CAPN13 NM 144575 calpain 13 CAPN3 NM 212464 calpain 3 isoform g CAPN7 NM O14296 calpain 7 CAPS2 NM 032606 calcyphosphine 2 CARD10 NM 014550 caspase recruitment domain protein 10 CARD14 NM 052819 caspase recruitment domain protein 14 isoform 2 CARD4 NM OO6092 caspase recruitment domain family, member 4 NM O14959 caspase recruitment domain family, member 8 NM 013276 carbohydrate kinase-like NM OO7359 cancer Susceptibility candidate 3 NM 138423 cancer Susceptibility candidate 4 isoform a NM 032982 caspase 2 isoform 1 preproprotein NM 001226 caspase 6 isoform alpha preproprotein NM OO1227 caspase 7 isoform alpha precursor NM OO1228 caspase 8 isoform A NM 172097 Sperm-associated cation channel 2 isoform 4 NM OO1753 caveolin 1 NM OO1233 caveolin 2 isoform a and b NM OO6807 chromobox homolog 1 (HP1 beta homolog Drosophila NM 005189 chromobox homolog 2 isoform 1 NM 175709 chromobox homolog 7 NM O17721 putative NFkB activating protein NM 032449 Coiled-coil and C2 domain containing 1B NM 133459 collagen and calcium binding EGF domains 1 CCBL1 NM OO4059 cytoplasmic conjugate-beta lyase CCDC14 NM O22757 Coiled-coil domain containing 14 CCDC15 NM O25004 Coiled-coil domain containing 15 CCDC16 NM 052857 Coiled-coil domain containing 16 CCDC25 NM OO 1031708 Coiled-coil domain containing 25 isoform 1 CCDC43 NM 144609 hypothetical protein LOC124808 CCDC52 NM 144718 hypothetical protein LOC152185 CCDC6 NM 005436 Coiled-coil domain containing 6 US 2009/0163434 A1 Jun. 25, 2009 18

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description CCDC68 NM O25214 CTCL tumor antigen se57-1 CCDC69 NM O15621 hypothetical protein LOC26112 CCL1 NM OO2981 Small inducible cytokine A1 precursor CCL28 NM 019846 Small inducible cytokine A28 precursor CCL5 NM OO2985 Small inducible cytokine A5 precursor CCND1 NM 053056 cyclin D1 CCND2 NM OO1759 cyclin D2 CCNE2 NM 057735 cyclin E2 isoform 2 CCNF NM OO1761 cyclin F CCNG2 NM 004354 cyclin G2 CCNJ NM O19084 cyclin J CCNT2 NM OO1241 cyclin T2 isoform a CCR6 NM 004367 chemokine (C-C motif) receptor 6 CCRL1 NM O16557 chemokine (C-C motif) receptor-like 1 CCS NM OO5125 copper chaperone for Superoxide dismutase CD2OO NM 0010041.96 CD200 antigen isoform b CD28 NM OO6139 CD28 antigen CD3OOLG NM 145273 triggering receptor expressed on myeloid cells CD36 NM 000072 CD36 antigen CD38 NM OO1775 CD38 antigen CD46 NM 002389 CD46 antigen, complement regulatory protein CD47 NM 001025079 CD47 molecule isoform 3 precursor CD59 NM 000611 CD59 antigen p18-20 CD68 NM OO1251 CD68 antigen CD69 NM OO1781 CD69 antigen (p60, early T-cell activation CD82 NM 001024844 CD82 antigen isoform 2 CD84 NMOO3874 CD84 antigen (leukocyte antigen) CD96 NM OO5816 CD96 antigen isoform 2 precursor CD99L2 NM 031462 CD99 antigen-like 2 isoform E3'-E4'-E3-E4 CDAN1 NM 138477 codanin 1 CDC23 NM 004661 cell division cycle protein 23 CDC37L1 NM O17913 cell division cycle 37 homolog (S. CDC4O NM O15891 pre-mRNA splicing factor 17 CDC42SE1 NM O2O239 CDC42 Small effector 1 CDCA4 NM O17955 cell division cycle associated 4 CDCA7 NM 031942 cell division cycle associated protein 7 isoform CDH2O NM 031891 cadherin 20, type 2 preproprotein CDK2AP2 NM OO5851 CDK2-associated protein 2 CDK5R1 NM OO3885 cyclin-dependent kinase 5, regulatory subunit 1 CDK6 NM OO1259 cyclin-dependent kinase 6 CDKN1A NM 000389 cyclin-dependent kinase inhibitor 1A CDT1 NM 030928 DNA replication factor C NM 031890 cat eye syndrome chromosome region, candidate 6 NM O16174 cerebral endothelial cell adhesion molecule 1 C NM OO1408 cadherin EGF LAG seven-pass G-type receptor 2 C NM 016343 protein F (350/400 kD) C NM 018.404 centaurin-alpha 2 protein C NM 012287 centaurin, beta 2 C NM O15230 centaurin delta 1 isoform a CEP135 NM O25009 centrosome protein 4 CEP152 NM O14985 hypothetical protein LOC22995 CEP170 NM O14812 centrosomal protein 170 kDa CEP27 NM 018097 hypothetical protein LOC55142 CEP57 NM 014679 Translokin CEP70 NM 024491 centrosomal protein 70 kDa CERK NM O22766 ceramide kinase isoform a CES2 NM OO3869 carboxylesterase 2 isoform 1 CETN2 NM 004344 Caltractin CFL2 NM 021914 cofilin 2 CFLAR NM OO3879 CASP8 and FADD-like apoptosis regulator CGNL1 NM 032866 cingulin-like 1 CHAF1A NM 005483 chromatin assembly factor 1, subunit A (p150) CHDS NM O15557 chromodomain helicase DNA binding protein 5 CHD6 NM 032221 chromodomain helicase DNA binding protein 6 CHD9 NM O25134 chromodomain helicase DNA binding protein 9 CHES1 NM 005197 checkpoint Suppressor 1 ChOn NM 018371 chondroitin beta1,4 CHML NM OO1821 choroideremia-like Rab escort protein 2 CHMP4C NM 152284 chromatin modifying protein 4C CHRFAM7A NM 1393.20 CHRNA7-FAM7A fusion isoform 1 US 2009/0163434 A1 Jun. 25, 2009 19

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Transcript ID (Pruitt et al., 2005) Description NM OOO739 cholinergic receptor, muscarinic 2 NM OOO745 cholinergic receptor, nicotinic, alpha NM OOO746 cholinergic receptor, nicotinic, alpha 7 NM OOO747 nicotinic acetylcholine receptor beta 1 subunit NM 000750 cholinergic receptor, nicotinic, beta NM 021615 carbohydrate (N-acetylglucosamine 6-O) NM O14918 carbohydrate (chondroitin) synthase 1 NM 145165 churchill domain containing 1 NM O2O313 cytokine induced apoptosis inhibitor 1 NM 004.895 cryopyrin isoform a NM O15125 capicua homolog NM 007174 Citron NM 133467 Cbp/p300-interacting transactivator, with NM 018204 cytoskeleton associated protein 2 NM O15282 CLIP-associating protein 1 NM 001286 chloride channel 6 isoform CC-6a NM OO56O2 claudin 11 NM 012129 claudin 12 NM 138429 claudin 15 isoform 2 NM OO1002026 claudin 18 isoform 2 NM 148960 claudin 19 NM O2O384 claudin 2 NM 019895 hypothetical protein LOC56650 NM 205852 macrophage antigenh NM 001004419 Osteoclast inhibitory lectin isoform 2 NM 080387 C-type lectin domain family 4, member D NMO13943 chloride intracellular channel 4 NM 016929 chloride intracellular channel 5 NM OO6493 ceroid-lipofuscinosis, neuronal 5 NM 018941 CLN8 protein NM 004898 Clock NM 001009566 calsyntenin 1 isoform 1 NM 0221.31 calsyntenin 2 NM 016308 cytidylate kinase NM 178818 chemokine-like factor Superfamily 4 isoform 1 NM O17801 CKLF-like MARVEL transmembrane domain containing NM O14865 chromosome condensation-related SMC-associated NM 018235 CNDP dipeptidase 2 (metallopeptidase M20 NM O19098 cyclic nucleotide gated channel beta 3 NM 001299 calponin 1, basic, Smooth muscle NM 199077 cyclin M2 isoform 3 NM O17623 cyclin M3 isoform 1 NM OO10O8225 CCR4-NOT transcription complex, Subunit 4 NM O15455 CCR4-NOT transcription complex, Subunit 6 NM 013354 CCR4-NOT transcription complex, Subunit 7 NM 016083 central cannabinoid receptor isoform a NM 000614 ciliary neurotrophic factor NM 020872 contactin 3 NM 014141 cell recognition molecule Caspir2 precursor NM 03.3655 cell recognition molecule CASPR3 NM O15198 cordon-bleu homolog NM 031431 component of golgi transport complex 3 NM 153603 component of oligomeric golgi complex 7 COIL NM 004645 COL11A2 NM 080679 collagen, type XI, alpha 2 isoform 3 COL19A1 NM OO1858 alpha 1 type XIX collagen precursor COL4A1 NM OO1845 alpha 1 type IV collagen preproprotein COL4A2 NM 001846 alpha 2 type IV collagen preproprotein COL4A3 NM 000091 alpha 3 type IV collagen isoform 1 precursor COL4A4 NM 000092 alpha 4 type IV collagen precursor COL8A2 NM O05202 collagen, type VIII, alpha 2 COLEC12 NM 030781 collectin sub-family member 12 isoform II COLO NM OO5677 acetylcholinesterase collagen-like tail Subunit COMMD10 NM 016144 COMM domain containing 10 COMMD2 NM 016094 COMM domain containing 2 COMMD4 NM 017828 COMM domain containing 4 COMMDS NM 014066 hypertension-related calcium-regulated gene COPA NM 004371 coatomer protein complex, Subunit alpha COPS6 NM OO6833 COP9 signalosome subunit 6 COQ2 NM O15697 para-hydroxybenzoate-polyprenyltransferase, US 2009/0163434 A1 Jun. 25, 2009 20

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description COQ7 NM O16138 COQ7 protein CORIN NM OO6587 Corin CORO1C NM O14325 coronin, actin binding protein, 1C CORO2B NM OO6091 coronin, actin binding protein, 2B COX6B2 NM 144613 cytochrome c oxidase subunit VIb, COX7A2L NM 004718 cytochrome c oxidase subunit VIIa polypeptide 2 COX8C NM 182971 cytochrome c oxidase subunit 8C P110 NM 014711 CP110 protein PEB3 NM O14912 cytoplasmic polyadenylation element binding PM NM 0010055O2 carboxypeptidase M precursor PNE1 NM OO3915 copine I POX NM OOOO97 coproporphyrinogen oxidase PS1 NM OO1875 carbamoyl-phosphate synthetase 1, mitochondrial NM 007007 cleavage and polyadenylation specific factor 6, R NM 000573 complement receptor 1 isoform F precursor RAMP1L, NM 020825 Crm, cramped-like EB1 NM 004379 cAMP responsive element binding protein 1 EB5 NM 001011666 cAMP responsive element binding protein 5 EBL2 NM 001310 cAMP responsive element binding protein-like 2 EM NM 181571 cAMP responsive element modulator isoform a M1 NM 016441 cysteine-rich motor neuron 1 NM 014171 postsynaptic protein CRIPT NM OO52O6 v-crk sarcoma virus CT10 oncogene homolog 1 NM 001014809 collapsin response mediator protein 1 isoform 1 sR NM 021151 carnitine O-octanoyltransferase NM OOO567 C-reactive protein, pentraxin-related NMOO4830 cofactor required for S p1 transcriptional NM 004268 cofactor required for S p1 transcriptional NM 004831 cofactor required for S p1 transcriptional NM OO4270 cofactor required for S p1 transcriptional NM 019604 class-I MHC-restricte T cell associated 117 cryptochrome 2 (photo lyase-like) NM 004077 citrate synthase precursor, isoform a NM O15989 cysteine Sulfinic acid decarboxylase-related NM OO1.007553 upstream of NRAS isoform 1 NM OO6140 colony stimulating factor 2 receptor alpha chain NM 052896 CUB and Sushi multiple domains 2 CSNK1 G1 NM 001011664 casein kinase 1, gamma 1 isoform L. CSNK2A1 NM OO1895 casein kinase II alpha 1 subunit isoform a CSTF2T NM O15235 cleavage stimulation factor, 3' pre-RNA, subunit CTAGE1 NM 022663 cutaneous T-cell lymphoma-associated antigen 1 CTDSPL NM OO1008392 Small CTD phosphatase 3 isoform 1 CTDSPL2 NM 016396 CTD (carboxy-terminal domain, RNA polymerase II, CTF NM OO1330 cardiotrophin 1 CTNND1 NM OO1331 catenin (cadherin-associated protein), delta 1 CTNS NM OO1031681 cystinosis, nephropathic isoform 1 CTSB NM OO1908 cathepsin B preproprotein CTSC NM 148170 cathepsin C isoform b precursor CTSK NM OOO396 cathepsin K preproprotein CTSS NM 004079 cathepsin Spreproprotein CTTNBP2NL NM 018704 hypothetical protein LOC55917 CUBN NM 001081 Cubilin CUGBP2 NM 001025076 CUG triplet repeat, RNA binding protein 2 CUL1 NM 003592 cullin 1 CUL3 NM 003590 cullin 3 CUTL2 NM O15267 cut-like 2 CX3CL1 NM OO2996 chemokine (C-X3—C motif) ligand 1 CX40.1 NM 153368 connexin40.1 CXCL14 NM OO4887 Small inducible cytokine B14 precursor CXCLS NM OO2994 chemokine (C X-C motif) ligand 5 precursor CXCL6 NM OO2993 chemokine (C X-C motif) ligand 6 (granulocyte CXCL9 NM 002416 Small inducible cytokine B9 precursor CXorf2O NM 153346 hypothetical protein LOC139105 CXorf21 NM O251.59 hypothetical protein LOC80231 CXorf38 NM 144970 hypothetical protein LOC159013 CXorf241 NM 173494 hypothetical protein LOC139212 CXorf53 NM 001018055 BRCA1/BRCA2-containing complex subunit 36 CXorf6 NM OO5491 hypothetical protein LOC10046 CXXC6 NM 03.0625 CXXC finger 6 CYB561D1 NM 182580 cytochrome b-561 domain containing 1 US 2009/0163434 A1 Jun. 25, 2009 21

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description CYB5B NM O30579 cytochrome b5 outer mitochondrial membrane CYB5D1 NM 144607 hypothetical protein LOC124637 CYBB NM OOO397 cytochrome b-245, beta polypeptide (chronic CYBRD1 NM 024843 cytochrome b reductase 1 CYCS NM 018947 cytochromec CYLD NM O15247 ubiquitin carboxyl-terminal hydrolase CYLD CYLN2 NM OO3388 cytoplasmic linker 2 isoform 1 CYP19A1 NM OOO103 cytochrome P450, family 19 CYP26B1 NM O19885 cytochrome P450, family 26, subfamily b, CYP2U1 NM 183075 cytochrome P450, family 2, Subfamily U, CYP2W1 NM O17781 cytochrome P450, family 2, Subfamily W. CYP4F3 NM OOO896 cytochrome P450, family 4, Subfamily F, CYSLTR2 NM O2O377 cysteinyl leukotriene receptor 2 D21S2O56E NM OO3683 nucleolar protein NOP52 DAPK2 NM O14326 eath-associated protein kinase 2 DAZAP2 NM 014764 DAZ associated protein 2 DBF4 NM OO6716 activator of S phase kinase DBF4B NM O25104 DBF4 homolog B isoform 2 DBT NM OO1918 ihydrolipoamide branched chain transacylase DCBLD2 NM 080927 iscoidin, CUB and LCCL domain containing 2 DCLRE1C NM OO 1033855 artemis protein isoform a DCTN4 NM 016221 ynactin 4 (p62) DCTNS NM 032486 ynactin 4 DCUN1D3 NM 173475 hypothetical protein LOC123879 DCUN1D4 NM O15115 DCN1, defective in cullin neddylation 1, domain DAH NM 012137 imethylarginine dimethylaminohydrolase 1 DB2 NMOOO107 amage-specific DNA binding protein 2 (48 kD) DHD NM 030637 DDHD domain containing 1 DHD2 NM O15214 DDHD domain containing 2 DOST NM OO5216 olichyl-diphosphooligosaccharide-protein DX1 NM 030655 DEAD H (Asp-Glu-Ala-Asp/His) box polypeptide 11 DX2 NM OO4728 DEAD (Asp-Glu-Ala-Asp) box polypeptide 21 DX26B NM 182540 hypothetical protein LOC203522 DX46 NM O14829 DEAD (Asp-Glu-Ala-Asp) box polypeptide 46 DX5 NM 004396 DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 DX5 NM 175066 DEAD (Asp-Glu-Ala-Asp) box polypeptide 51 DX55 NM 020936 DEAD (Asp-Glu-Ala-Asp) box polypeptide 55 DX59 NM 031306 DEAD (Asp-Glu-Ala-Asp) box polypeptide 59 EADC1 NM 182SO3 deaminase domain containing 1 EAF NM 021008 Suppressin ECR2 NM 020664 2,4-dienoyl CoA reductase 2, peroxisomal EDD NM 032998 death effector domain-containing protein EFB106A NM 152251 defensin, beta 106A EGS NM OO3676 degenerative spermatocyte homolog 1, lipid ENND1A NM 024820 hypothetical protein LOC57706 isoform 2 ENND2C NM 198459 DENN/MADD domain containing 2C ENND3 NM O14957 hypothetical protein LOC22898 ENND4C NM O17925 hypothetical protein LOC55667 EPDC4 NM 152317 DEP domain containing 4 ERL2 NM 016041 Derl-like domain family, member 2 FFA NM 004401 DNA fragmentation factor, 45 kDa, alpha FNAS NM 004403 eafness, autosomal dominant 5 protein GAT2L4 NM OO10O2254 iacylglycerol O-acyltransferase 2-like 4 GCR13 NM 001024733 DiGeorge syndrome gene H NM OO1347 iacylglycerol kinase, theta HDDS NM O24887 ehydrodolichyl diphosphate synthase isoform a HFRL1 NM 176815 hypothetical protein LOC200895 HODH NM 0010251.93 ihydroorotate dehydrogenase isoform 2 HTKD1 NM 0187O6 ehydrogenase E1 and transketolase domain NM 014681 DEAH (Asp-Glu-Ala-His) box polypeptide 34 CER1 NM 030621 icer DO1 NM 033081 eath inducer-obliterator 1 isoform c NM OOO792 eiodinase, iodothyronine, type I isoform a NM O15151 DIP2-like protein isoform a NM 173602 hypothetical protein LOC576.09 RC1 NM 052952 hypothetical protein LOC116093 SC1 NM 001012957 isrupted in schizophrenia 1 isoform LV NM 033425 DIX domain containing 1 isoform b NM O15395 hypothetical protein LOC25851 NM 194295 hypothetical protein LOC196968 US 2009/0163434 A1 Jun. 25, 2009 22

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description NM 001003.399 hypothetical protein LOC400169 NM O15459 hypothetical protein LOC25923 NM 0321.21 implantation-associated protein NM 207323 hypothetical protein LOC147172 NM 033107 claudin 12 NM 194282 hypothetical protein LOC132660 NM 0010099.13 hypothetical protein LOC374383 NM 138368 hypothetical protein LOC91056 NM 152411 hypothetical protein LOC136051 NM 152622 hypothetical protein LOC166968 NM OO6094 deleted in liver cancer 1 isoform 2 DLEC1 NM 005106 deleted in lung and esophageal cancer 1 isoform DLGAP2 NM 004745 discs large-associated protein 2 NM OO5221 distal-less homeobox 5 DMBX1 NM 147192 diencephalon mesencephalon homeobox 1 isoform b DMC NM 007068 DMC1 dosage Suppressor of mck1 homolog DMN NM O15286 desmuslin isoform B DMP NM 004.407 dentin matrix acidic phosphoprotein DMRT2 NM OO6557 doublesex and mab-3 related transcription factor DMTF1 NM 021145 cyclin D binding myb-like transcription factor DNAJA4 NM 018602 DnaJ (Hsp40) homolog, subfamily A, member 4 DNAJAS NM 00101.2339 DnaJ homology subfamily A member 5 isoform 2 DNAB6 NM OO5494 DnaJ (Hsp40) homolog, subfamily B, member 6 DNAB9 NM 012328 DnaJ (Hsp40) homolog, subfamily B, member 9 DNAJC15 NM 013238 DNAJ domain-containing DNAJC18 NM 152686 DnaJ (Hsp40) homolog, subfamily C, member 18 DNAJC19 NM 145261 translocase of the inner mitochondrial membrane DNAJCS NM O25219 DnaJ (Hsp40) homolog, subfamily C, member 5 DNASE2 NM OO1375 eoxyribonuclease II, lysosomal precursor DNM2 NM 001005360 ynamin 2 isoform 1 DNM3 NM O15569 ynamin 3 DOCK9 NM O15296 edicator of cytokinesis 9 DOPEY2 NM O05128 paid-1-like DPP10 NM OO1004360 ipeptidyl peptidase 10 isoform short DPP3 NM 005700 ipeptidyl peptidase III DPP9 NM 139159 ipeptidylpeptidase 9 DPY19L3 NM 207325 py-19-like 3 DPYD NM 000110 ihydropyrimidine dehydrogenase DPYSL5 NM 020134 ihydropyrimidinase-like 5 DRD1 NM 000794 opamine receptor D1 DSC3 NM 024423 esmocollin 3 isoform Dsc3b preproprotein DSG4 NM 177986 esmoglein 4 DSPG3 NM OO4950 ermatan Sulfate proteoglycan 3 precursor DTWD2 NM 173666 DTW domain containing 2 DUSP10 NM OO72O7 ual specificity phosphatase 10 isoform a DUSP13 NM OO1007271 muscle-restricted dual specificity phosphatase DUSP18 NM 152511 ual specificity phosphatase 18 DUSP2 NM 004418 ual specificity phosphatase 2 DUSP6 NM OO1946 ual specificity phosphatase 6 isoform a DUSP8 NM 004420 ual specificity phosphatase 8 DUXA NM 001012729 hypothetical protein LOC503835 DVL3 NM 004423 ishevelled 3 DXS98.79E NM OO6014 ESO3 protein DYNC1LI2 NM 006141 ynein, cytoplasmic, light intermediate DYNLT1 NM OO6519 -complex-associated-testis-expressed 1-like 1 DYRK1A NM 001396 ual-specificity tyrosine-(Y)-phosphorylation DYRK2 NM 003583 ual-specificity tyrosine-(Y)-phosphorylation DZIP1 NM O14934 DAZ interacting protein 1 isoform 1 E2F1 NM OO5225 E2F transcription factor 1 NM 004.091 E2F transcription factor 2 NM OO1949 E2F transcription factor 3 NM OO1951 E2F transcription factor 5 EAF1 NM 033083 ELL associated factor 1 NM 001005463 early B-cell factor 3 EBI2 NM 004.951 EBV-induced G protein-coupled receptor 2 EDA NM 001005610 ectodysplasin A isoform EDA-B EDA2R NM 021783 X-linked ectodysplasin receptor EDD1 NM O15902 E3 ubiquitin protein ligase, HECT domain EDEM1 NM 014674 ER degradation enhancer, mannosidase alpha-like EDG1 NM OO1400 endothelial differentiation, sphingolipid US 2009/0163434 A1 Jun. 25, 2009 23

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Transcript ID (Pruitt et al., 2005) Description NM OO5226 endothelial differentiation, sphingolipid NM 181723 EF hand domain family, member A2 NM 004428 ephrin A1 isoform a precursor NM 004.429 ephrin-B1 precursor NM 004.093 ephrin B2 NM O24580 elongation factor Tu GTP binding domain NM 001410 EGF-like-domain, multiple 4 NM O22051 egl nine homolog 1 NM O22073 egl nine homolog 3 NM OOO399 early growth response 2 protein NM 004430 early growth response 3 NM 014600 EH-domain containing 3 NM OO1966 enoyl-Coenzyme A, hydratase 3-hydroxyacyl NM O24757 euchromatic histone methyltransferase 1 NM OO1007277 etoposide induced 2.4 isoform 2 NM 152361 EID-2-like inhibitor of differentiation-3 NM 001013703 eukaryotic translation initiation factor 2 alpha NM 012199 eukaryotic translation initiation factor 2C, 1 NM 004.094 eukaryotic translation initiation factor 2, NM 003757 eukaryotic translation initiation factor 3, NM 004.096 eukaryotic translation initiation factor 4E NM 001418 eukaryotic translation initiation factor 4 NM OO1969 eukaryotic translation initiation factor 5 NM O2O390 eIF-5A2 protein NM OO5230 ELK3 protein NM 152793 hypothetical protein LOC222166 NMO14800 engulfment and cell motility 1 isoform 1 NM 018712 ELMO domain containing 1 NM 013447 egf-like module containing, mucin-like, hormone NM 004.098 empty spiracles homolog 2 NM OO1427 engrailed homolog 2 NM OO1008493 enabled homolog isoform a NM 031889 Enamelin NM OO1975 enolase 2 NM O14936 ectonucleotide pyrophosphatase/phosphodiesterase NM 021572 ectonucleotide pyrophosphatase/phosphodiesterase NM 2071.68 endosulfine alpha isoform 8 NM 004901 ectonucleoside triphosphate diphosphohydrolase NM OO1247 ectonucleoside triphosphate diphosphohydrolase NM 001430 endothelial PAS domain protein 1 NM OO4437 erythrocyte membrane protein band 4.1 NM 012156 erythrocyte membrane protein band 4.1-like 1 NM 001431 erythrocyte membrane protein band 4.1-like 2 NM 019114 erythrocyte membrane protein band 4.1 like 4B NM 020909 erythrocyte membrane protein band 4.1 like 5 NM 017549 upregulated in colorectal cancer gene 1 protein NM 004438 ephrin receptor EphA4 NM 004439 ephrin receptor EphA5 isoform a A.7 NM 004440 ephrin receptor EphA7 NM 004441 ephrin receptor EphB1 precursor NM 004444 ephrin receptor EphB4 precursor NM OO5670 laforin isoform a NM O14805 EPM2A interacting protein 1 NM OO1 OO6600 ERBB2 interacting protein isoform 7 B23A. NM OO1982 erbB-3 isoform 1 precursor EG NM OO1432 epiregulin precursor NM 004449 v-ets erythroblastosis virus E26 oncogene like NM 020462 endoplasmic reticulum-golgi intermediate NM 152461 endoplasmic reticulum to nucleus signalling 1 NM 019891 endoplasmic reticulum oxidoreductin 1-Lbeta NM OOO125 estrogen receptor 1 NM 024311 hypothetical protein LOC79157 E NM OO4730 eukaryotic translation termination factor 1 NM OO4956 ets variant gene 1 ETV5 NM 004.454 ets variant gene 5 (ets-related molecule) EVA NM OO5797 epithelial V-like antigen 1 precursor EXOSC1 NM 016046 exosomal core protein CSL4 EYA NM 000503 eyes absent 1 isoform b EYA4 NM 004100 eyes absent 4 isoform a EZH NM OO1991 enhancer of Zeste homolog 1 US 2009/0163434 A1 Jun. 25, 2009 24

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description NM 016946 F11 receptor isoform a precursor NM OO1992 coagulation factor II receptor precursor NM OO5242 coagulation factor II () receptor-like 1 NM 004101 coagulation factor II (thrombin) receptor-like 2 NM OO3950 coagulation factor II (thrombin) receptor-like 3 NM OO1993 coagulation factor III precursor NM OOO133 coagulation factor IX FADS1 NM 013402 atty acid desaturase 1 FADS6 NM 178128 atty acid desaturase domain family, member 6 FAHD1 NM 031208 umarylacetoacetate hydrolase domain containing FAIM2 NM 012306 Fas apoptotic inhibitory molecule 2 FAM102A NM 203305 early estrogen-induced gene 1 protein isoform b FAM106A NM O24974 hypothetical protein LOC80039 FAM107A NM 007177 downregulated in renal cell carcinoma FAM107B NM 031453 hypothetical protein LOC83641 FAM13A1 NM 001015045 amily with sequence similarity 13, member A1 FAM13C1 NM OO1OO1971 hypothetical protein LOC220965 isoform 2 FAM18B NM 016078 hypothetical protein LOC51030 FAM19A1 NM 213609 amily with sequence similarity 19 (chemokine FAM36A NM 198076 amily with sequence similarity 36, member A FAM3A NM O21806 amily 3, member A protein FAM3C NM O14888 predicted Osteoblast protein FAM40A NM 033088 hypothetical protein LOC85369 FAM4OB NM 020704 hypothetical protein LOC57464 FAM43A NM 153690 hypothetical protein LOC131583 FAM45A NM 207009 hypothetical protein LOC404636 FAM45B NMO18472 hypothetical protein LOC55855 FAM46C NM O17709 hypothetical protein LOC54855 FAM46D NM 152630 hypothetical protein LOC169966 FAMS3B NM 014661 hypothetical protein LOC9679 FAMS3C NM 016605 amily 53, member C protein FAMS4B NM O19557 hypothetical protein LOC56181 FAMSSC NM 145037 hypothetical protein LOC91775 FAMS7A NM O24792 amily with sequence similarity 57, member A FAM60A NM 021238 amily th equence similarity 60, member A FAM62B NM O2O728 amily with sequence similarity 62 (C2 domain FAM65A NM O24519 hypothetical protein LOC79567 FAM7OA NM O17938 hypothetical protein LOC55026 FAM73A NM 198549 hypothetical protein LOC374986 FAM73B NM 032809 hypothetical protein LOC84895 FAM79A NM 182752 hypothetical protein LOC127262 FAM79B NM 198485 hypothetical protein LOC285386 FAM82C NM 018145 amily with sequence similarity 82, member C FAM83D NM 030919 hypothetical protein LOC81610 FAM83E NM O17708 hypothetical protein LOC54854 FAM83H NM 198488 hypothetical protein LOC286.077 FAM84B NM 174911 breast cancer membrane protein 101 FAM86C NM 018172 hypothetical protein LOC55199 isoform 1 FAM8A1 NM O16255 Autosomal Highly Conserved Protein FAM98B NM 173611 hypothetical protein LOC283742 EANCC NM OOO136 Fanconi anemia, complementation group C EANCD2 NM 033084 Fanconi anemia complementation group D2 isoform FARSLB NM 005687 phenylalanine-tRNA synthetase-like, beta FASLG NM 000639 as ligand FASTK NM OO6712 Fas-activated serine/threonine kinase isoform 1 FAT2 NM OO1447 FAT tumor Suppressor 2 precursor FBLIM NM 001024216 filamin-binding LIM protein-1 isoform c FBLN1 NM OO6486 fibulin 1 isoform D FBN2 NM OO1999 fibrillin 2 precursor FBXL1 NM 012308 F-box and leucine-rich repeat protein 11 FBXL18 NM 024963 F-box and leucine-rich repeat protein 18 FBXL22 NM 203373 hypothetical protein LOC283807 FBXL3 NM 012158 F-box and leucine-rich repeat protein 3 FBXLS NM 012161 F-box and leucine-rich repeat protein 5 isoform FBXL7 NM 012304 F-box and leucine-rich repeat protein 7 FBXO11 NM O25133 F-box only protein 11 isoform 1 FBXO18 NM 032807 F-box only protein, helicase, 18 isoform 1 FBXO21 NM O15002 F-box only protein 21 isoform 2 FBXO27 NM 178820 F-box protein 27 FBXO31 NM O24735 F-box protein 31 US 2009/0163434 A1 Jun. 25, 2009 25

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description NM 153230 F-box protein 39 NM O16298 F-box protein 40 NM 018438 F-box only protein 6 NM 012347 F-box only protein 9 isoform 1 NM 012300 F-box and WD-40 domain protein 1B isoform C FC HO2 NM 138782 FCH domain only 2 FCMD NM OO6731 Fukutin FEM1C NM O2O177 eminization 1 homolog a FEZ2 NM 005102 Zygiin 2 NM 004463 aciogenital dysplasia protein NM 139241 FYVE, RhoGEF and PH domain containing 4 NM 152536 FYVE, RhoGEF and PH domain containing 5 NM OO2006 fibroblast growth factor 2 NM OO2007 fibroblast growth factor 4 precursor NM 004464 fibroblast growth factor 5 isoform 1 precursor F7 NM OO2009 fibroblast growth factor 7 precursor FR1 NM O23107 fibroblast growth factor receptor 1 isoform 5 FR2 NM 022973 fibroblast growth factor receptor 2 isoform 6 NM OO6682 fibrinogen-like 2 NM 022116 fidgetin-like 1 NM O14344 our jointed box 1 NM O17946 FK506 binding prote in 14, 22 kDa NM 004116 FK506-binding protein 1B isoform a NM OO4117 FK506 binding protein 5 NM 024301 ukutin-related protein NM 001014342 2 O159 NMO18013 hypothetical protein LOCSSO84 O241 NM 018035 hypothetical protein LOCSS101 O357 NM 018071 hypothetical protein LOC55701 O781 NM 018215 hypothetical protein LOCSS228 O803 NM 018224 hypothetical protein LOCSS744 O925 NM 018275 hypothetical protein LOCSS262 1021 NM 023012 hypothetical protein LOC65117 isoform a 1151 NM 018340 hypothetical protein LOC55313 1171 NM 018348 hypothetical protein LOC55.783 1259 NM 018370 hypothetical protein LOC55332 1292 NM 018382 hypothetical protein LOCSS338 1806 NM 024824 UKp68 isoform 1 2331 NM 024986 hypotnetical protein LOC8OOS2 2505 NM O24749 hypothetical protein LOC798OS 2949 NM 023008 hypothetical protein LOC65095 isoform 1 3236 NM 024902 hypothetical protein FLJ13236 3576 NM 022484 hypothetical protein LOC64418 3639 NM O24705 hypothetical protein FLJ13639 isoform 2 3646 NM 024584 hypothetical protein LOC79635 3841 NM O24702 hypothetical protein LOC79755 3946 NM 152275 hypothetical protein LOC92104 3984 NM O24770 hypothetical protein LOC79828 4107 NM O25026 hypothetical protein LOC80094 4213 NM 024841 hypothetical protein LOC798.99 4397 NM 032779 hypothetical protein LOC8486S 4437 NM 032578 Myopalladin 4466 NM 032790 hypothetical protein LOC84876 4503 NM 152780 hypothetical protein LOC2S6714 6008 NM 001001665 hypothetical protein LOC339761 6237 NM OO1004320 hypothetical protein LOC3926.36 6542 NM OO1004301 hypothetical protein LOC126O17 20032 NM O17628 hypothetical protein LOCS4790 2O186 NM 207514 differentially expressed in FDCP8 isoform 1 2O294 NM O17749 hypothetical protein LOCSS626 2O298 NM O17752 hypothetical protein LOC54885 isoform a 2O366 NM O17786 hypothetical protein FLJ2O366 204.87 NM O17841 hypothetical protein LOCS4949 20489 NM O17842 hypothetical protein LOC55652 20758 NM O17952 hypothetical protein LOC55037 20972 NM O25030 hypothetical protein LOC80098 2112S NM O24627 hypothetical protein LOC7968O 21657 NM 022483 hypothetical protein LOC64417 21687 NM O24859 PDZ domain containing, X chromosome 21736 NM 024922 esterase 31

US 2009/0163434 A1 Jun. 25, 2009 27

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description FLJ45337 NM 207465 hypothetical protein LOC400754 FLJ45422 NM 001004349 hypothetical protein LOC441140 FLJ45.645 NM 198557 hypothetical protein LOC375287 FLJ45909 NM 1984.45 hypothetical protein LOC126432 FLJ46247 NM 198529 hypothetical protein LOC374786 isoform 1 FLJ463.63 NM 207434 hypothetical protein LOC400002 FLJ46385 NM 001001675 hypothetical protein LOC390963 FLJ90013 NM 153365 hypothetical protein LOC202018 FLJ90396 NM 153358 hypothetical protein LOC163049 FLJ90579 NM 173591 hypothetical protein LOC283310 FLJ90757 NM OO1004336 hypothetical protein LOC440465 FLRT2 NM 013231 fibronectin leucine rich transmembrane protein FLT1 NM OO2019 ms-related tyrosine kinase 1 (vascular FLYWCH1 NM 032296 FLYWCH-type 1 isoform a FMNL2 NM OO1004417 ormin-like 2 isoform D FMNL3 NM 175736 ormin-like 3 isoform 1 FMO3 NM OO1.002294 flavin containing monooxygenase 3 isoform 2 FMOD NM 002023 fibromodulin precursor FNBP1 NM O15033 ormin binding protein 1 FNBP1L NM 001024948 ormin binding protein 1-like isoform 1 FNBP4. NM O15308 ormin binding protein 4 FNDC3A NM O14923 fibronectin type III domain containing 3A FNDC3B NM O22763 fibronectin type III domain containing 3B FNDC5 NM 153756 fibronectin type III domain containing 5 FOSL1 NM 005438 FOS-like antigen 1 FOXA1 NM 004496 orkhead box A1 FOXF1 NMOO1451 orkhead box F1 FOXJ2 NM 018416 orkhead box J2 FOXJ3 NM O14947 orkhead box J3 FOXL2 NM 023067 orkhead box L2 FOXO1 NM 033260 orkhead box Q1 PGT NM OO3838 lucose-1-phosphate guanyltransferase RAT2 NM 012083 GSK-3 binding protein FRAT2 REQ NM O14286 requenin homolog RMD4A NM 018027 FERM domain containing 4A RMD6 NM 152330 FERM domain containing 6 TS NM 00101.2398 used toes homolog UBP1 NM OO3902 air upstream element-binding protein UCA2 NM 032020 lucosidase, alpha-L-2, plasma UNDC2 NM O23934 FUN14 domain containing 2 URIN NM OO2569 urin preproprotein USIP1 NM 054O16 FUS interacting protein (serine-arginine rich) 1 UT2 NM 000511 lucosyltransferase 2 (secretor status included) UT3 NM OOO149 lucosyltransferase 3 (galactoside UT4 NM 002033 lucosyltransferase 4 UT5 NM 002034 lucosyltransferase 5 UT6 NM OOO150 lucosyltransferase 6 (alpha (1,3) FXN NM OOO144 rataxin isoform 1 preproprotein FXR1 NM 001013438 ragile X mental retardation-related protein 1 FXYD6 NM O22003 FXYD domain-containing ion transport regulator FYCO1 NM O24513 FYVE and coiled-coil domain containing 1 FZD10 NM 007197 rizzled 10 FZD4 NM 0121.93 rizzled 4 FZD6 NM 003506 rizzled 6 FZD7 NM 003507 rizzled 7 GAB1 NM 002039 GRB2-associated binding protein 1 isoform b GAB2 NM 012296 GRB2-associated binding protein 2 isoform b GAB3 NM 080612 Gab3 protein GABBR1 NM OO1470 gamma-aminobutyric acid (GABA) B receptor 1 GABBR2 NM 005458 G protein-coupled receptor 51 GABPB2 NM OO5254 GA binding protein transcription factor, beta GABRE NM 004961 gamma-aminobutyric acid (GABA) A receptor, GABRG1 NM 173536 gamma-aminobutyric acid A receptor, gamma 1 GABRG2 NM 198904 gamma-aminobutyric acid A receptor, gamma 2 GAK NM OO5255 cyclin G associated kinase GALIG NM 194327 galectin-3 internal gene GALK2 NM 001001556 galactokinase 2 isoform 2 GALM NM 1388O1 galactose mutarotase (aldose 1-epimerase) GALNT3 NM 004482 polypeptide N-acetylgalactosaminyltransferase 3 GALNT4 NM 003774 polypeptide N-acetylgalactosaminyltransferase 4 US 2009/0163434 A1 Jun. 25, 2009 28

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description GALNT6 NM 007210 polypeptide N-acetylgalactosaminyltransferase 6 GALNTL2 NM 054110 UDP-N-acetyl-alpha-D-galactosamine:polypeptide GAN NM 02204 Gigaxonin GARS NM 002047 glycyl-tRNA synthetase GAS7 NM 003644 growth arrest-specific 7 isoform a GAS8 NM 00148 growth arrest-specific 8 GATA6 NM OO5257 GATA binding protein 6 GATAD1 NM 021167 GATA zinc finger domain containing 1 GATS NM 17883 opposite strand transcription unit to STAG3 GBF1 NM OO4193 golgi-specific brefeldin A resistance factor 1 GBP1 NM 002053 guanylate binding protein 1, GBP3 NM 018284 guanylate binding protein 3 GBP4. NM 05294 guanylate binding protein 4 GCC2 NM 014635 GRIP and coiled-coil domain-containing 2 isoform GCET2 NM OO10O8756 germinal center expressed transcript 2 isoform GCLM NM 00206 glutamate-cysteine ligase regulatory protein GCNT2 NM OO149 glucosaminyl (N-acetyl) transferase 2, GCNT4 NM O1659 core 2 beta-1,6-N-acetylglucosaminyltransferase Gcom1 NM 001018100 GRINL1A upstream protein isoform 7 GDA NM OO4293 guanine deaminase GDPD1 NM 182569 glycerophosphodiester phosphodiesterase domain GEMINT NM OO1007269 gemin 7 GENX-3414 NM OO3943 genethonin 1 GFER NM OO5262 erv1-like growth factor GGA1 NM 001001561 golgi associated, gamma adaptin ear containing, GGT6 NM 153338 gamma-glutamyltransferase 6 homolog GIMAP8 NM 175571 GTPase, IMAP family member 8 GIOT-1 NM 153257 gonadotropin inducible transcription repressor GIPC2 NM O17655 PDZ domain protein GIPC2 GIT2 NM 014776 G protein-coupled receptor kinase-interactor 2 GA1 NM OOO165 connexin 43 GB7 NM 198568 hypothetical protein LOC375519 GKAP1 NM O25211 G kinase anchoring protein 1 GLB1L, NM 024506 galactosidase, beta 1-like GLDN NM 181789 Colomin GLO1 NM OO6708 glyoxalase I GLT2SD2 NM O15101 glycosyltransferase 25 domain containing 2 GLTP NM 016433 glycolipid transfer protein GM632 NM 020713 hypothetical protein LOC57473 GMCL1 NM 178439 germ cell-less GMCL1L. NM O22471 germ cell-less homolog 1 (Drosophila)-like GMFB NM 004124 glia maturation factor, beta GNAI1 NM 002069 guanine nucleotide binding protein (G protein), GNAZ NM 002073 guanine nucleotide binding protein, alpha Z GNBS NM OO6578 guanine nucleotide-binding protein, beta-5 GNE NM 005476 UDP-N-acetylglucosamine-2-epimerase/N- GNPDA2 NM 138335 glucosamine-6-phosphate deaminase 2 GNPNAT1 NM 198066 glucosamine-phosphate N-acetyltransferase 1 GNPTAB NM 024312 N-acetylglucosamine-1-phosphate transferase GNS NM OO2O76 glucosamine (N-acetyl)-6-sulfatase precursor GOLGA1 NM 002077 golgin 97 GOLGA2 NM 004486 Golgi autoantigen, golgin subfamily a 2 GOLPH2 NM O16548 golgi phosphoprotein 2 GOLPH3 NM 0221.30 golgi phosphoprotein 3 GORASP1 NM 031899 Golgi reassembly stacking protein 1 GOSR1 NM OO1007024 golgi SNAP receptor complex member 1 isoform 3 GP5 NM 004488 glycoprotein V (platelet) GPAM NM 020918 mitochondrial glycerol 3-phosphate GPATC2 NM 018040 G patch domain containing 2 GPD NM OO5276 glycerol-3-phosphate dehydrogenase 1 (Soluble) GPIAP1 NM OO5898 membrane component Surface marker GPR NM OO5279 G protein-coupled receptor 1 GPR114 NM 153837 G-protein coupled receptor 114 GPR126 NM OO1032394 G protein-coupled receptor 126 alpha 2 GPR132 NM 013345 G protein-coupled receptor 132 GPR135 NM O22571 G protein-coupled receptor 135 GPR137B NM OO3272 transmembrane 7 superfamily member 1 GPR155 NM OO1033045 G protein-coupled receptor 155 GPR176 NM OO7223 putative G protein coupled receptor GPR18O NM 180989 G protein-coupled receptor 180 precursor US 2009/0163434 A1 Jun. 25, 2009 29

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description GPR26 NM 153442 G protein-coupled receptor 26 GPR3 NM OO5281 G protein-coupled receptor 3 GPR37 NM OO5302 G protein-coupled receptor 37 GPR45 NM OO7227 G protein-coupled receptor 45 GPR6 NM OO5284 G protein-coupled receptor 6 GPR81 NM 032554 G protein-coupled receptor 81 GPR83 NM O16540 G protein-coupled receptor 83 GPR85 NM 018970 G protein-coupled receptor 85 GRAMD1A NM 020895 hypothetical protein LOC57655 GRB2 NM OO2O86 growth factor receptor-bound protein 2 isoform GREB1 NM 148903 GREB1 protein isoform c GRHL2 NM O24915 transcription factor CP2-like 3 GRIN3A NM 133445 glutamate receptor, ionotropic, GRIPAP1 NM 207672 GRIP1 associated protein 1 isoform 2 GRM1 NM 000838 glutamate receptor, metabotropic 1 GRM6 NM OOO843 glutamate receptor, metabotropic 6 precursor GRM7 NM OOO844 glutamate receptor, metabotropic 7 isoform a GRPEL2 NM 1524O7 GrpE-like 2, mitochondrial GRTP1 NM O24719 growth hormone regulated TBC protein 1 GSTM3 NM OOO849 glutathione S-transferase M3 GTDC1 NM OO1OO6636 glycosyltransferase-like domain containing 1 GTF2H2 NM OO1515 general transcription factor IIH, polypeptide 2, GTPBP5 NM O15666 GTP binding protein 5 GUCA1B NM O02098 guanylate cyclase activator 1B (retina) GUCY1A3 NM OOO856 guanylate cyclase 1, Soluble, alpha 3 GUCY1B2 NM 004129 guanylate cyclase 1, Soluble, beta 2 GYS1 NM002103 glycogen synthase 1 (muscle) H2AF NM 018267 H2A histone family, member J isoform 1 H2AFY2 NM 018649 core histone macroH2A2.2 H6PD NM OO4285 hexose-6-phosphate dehydrogenase precursor HARS NM 002109 istidyl-tRNA synthetase HBP1 NM 012257 HMG-box transcription factor 1 HBS1L NM O06620 HBS1-like HBXIP NM OO6402 hepatitis B virus X-interacting protein HCCS NM 005333 holocytochrome c synthase (cytochrome c HCLS1 NM 005335 ematopoietic cell-specific Lyn Substrate 1 HCP5 NM OO6674 HLA complex P5 HDAC4 NM OO6037 histone deacetylase 4 HDCMA18P NM 016648 hypothetical protein LOC51574 NM 012080 haloacid dehalogenase-like hydrolase domain NM O16217 Headcase NM 182765 HECT domain containing 2 isoform a NM O16173 HemK methyltransferase family member 1 NM O22373 hypothetical protein LOC64224 NM 207582 HERV-FRD provirus ancestral Env polyprotein NM O19089 hairy and enhancer of split homolog 2 NM 012259 hairyienhancer-of-split related with YRPW motif NM 033055 hippocampus abundant transcript 1 NM O15094 hypermethylated in cancer 2 NM OO1530 hypoxia-inducible factor 1, alpha Subunit NM 013332 hypoxia-inducible protein 2 NM 005338 huntingtin interacting protein 1 HIP1R NM OO3959 huntingtin interacting protein-1-related HIST1H2AG NM 021064 H2A histone family, member P HK1 NM 000188 hexokinase 1 isoform HKI HLA-DOA NM 002119 major histocompatibility complex, class II, DO HLCS NM 000411 holocarboxylase synthetase HLF NM 002126 hepatic leukemia factor HM13 NM 178582 minor histocompatibility antigen 13 isoform 4 HMGA2 NM 001015886 high mobility group AT-hook 2 isoform c HMGB3 NM OO5342 high-mobility group box 3 HMGCLL1 NM O19036 3-hydroxymethyl-3-methylglutaryl-Coenzyme A HMGN4 NM OO6353 high mobility group nucleosomal binding domain HMOX1 NM 002133 heme oxygenase (decyclizing) 1 HN1 NM OO1002032 hematological and neurological expressed 1 HNF4G NM 004.133 hepatocyte nuclear factor 4, gamma HNMT NM OO6895 histamine N-methyltransferase isoform 1 HNRPH2 NM OO1032393 heterogeneous nuclear ribonucleoprotein H2 HNRPU NM OO45O1 heterogeneous nuclear ribonucleoprotein U HNT NM O16522 Neurotrimin US 2009/0163434 A1 Jun. 25, 2009 30

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description HOOK3 NM 032410 golgi-associated microtubule-binding protein HOXA3 NM 030661 homeobox A3 isoform a OXB13 NM OO6361 homeobox B13 HOXB4 NM 024015 homeobox B4 P1BP3 NM O16287 HPCAL4 NM O16257 hippocalcin-like protein 4 HPGD NM OOO860 hydroxyprostaglandin dehydrogenase 15-(NAD) HPS5 NM 007216 Hermansky-Pudlak syndrome 5 isoform b HRB NM 004.504 HIV-1 Rev binding protein HRB2 NM 007043 HIV-1 rev binding protein 2 HRBL NM OO6076 HIV-1 Rev-binding protein-like protein HRH2 NM 022304 histamine receptor H2 HRH4 NM 021624 histamine H4 receptor S2ST1 NM 012262 heparan sulfate 2-O-sulfotransferase 1 S3ST4 NM 006040 heparan Sulfate D-glucosaminyl HSC2O NM 172002 -type co-chaperone HSC20 HSD17B7 NM 016371 hydroxysteroid (17-beta) dehydrogenase 7 PA5 NM OO5347 heat shock 70 kDa protein 5 (glucose-regulated PA6 NM 002155 heat shock 70 kDa protein 6 (HSP7OB') PA8 NM OO6597 heat shock 70 kDa protein 8 isoform 1 PCO47 NM 014147 hypothetical protein LOC29060 PCO65 NM 014157 hypothetical protein LOC29070 PC268 NM 197964 hypothetical protein LOC154791 NM OO6644 heat shock 105 kD NM OOO621 5-hydroxytryptamine (serotonin) receptor 2A NM 014586 hormonally upregulated Neu-associated kinase NMOO3549 hyaluronoglucosaminidase 3 RACs NM 016400 Huntingtin interacting protein K A. P P NM 000415 islet amyloid polypeptide precursor CAM4 NM 001544 intercellular adhesion molecule 4 isoform 1 NM 012405 isoprenylcysteine carboxyl methyltransferase FIT1 NM OO1548 interferon-induced protein with FIT3 NM OO1549 interferon-induced protein with FITS NM 012420 interferon-induced protein with FNAR1. NM 000629 interferon-alpha receptor 1 precursor FNAR2 NM 207585 interferon alpha/beta receptor 2 isoform a FRD2 NM OO6764 interferon-related developmental regulator 2 T8O NM 020800 WD repeat domain 56 F2BP1 NM OO6546 insulin-like growth factor 2 mRNA binding FBP5 NM 000599 insulin-like growth factor binding protein 5 FBP7 NM OO1553 insulin-like growth factor binding protein 7 FL3 NM 207393 insulin growth factor-like family member 3 PK1 NM OO1 OO6115 inositol hexaphosphate kinase 1 isoform 2 BKB NM OO1556 inhibitor of kappa light polypeptide gene NM 153687 IKK interacting protein isoform 1 O NM 000572 interleukin 10 precursor ORA NM OO1558 interleukin 10 receptor, alpha precursor NM 000641 interleukin 11 precursor NM OO1559 interleukin 12 receptor, beta 2 precursor NM O22789 interleukin 17E isoform 1 precursor NM 052872 interleukin 17F precursor NM 172234 interleukin 17B receptor isoform 2 precursor NM 017563 interleukin 17 receptor D NM 012275 interleukin 1 family, member 5 NM OOO877 interleukin 1 receptor, type I precursor NM 002182 interleukin 1 receptor accessory protein isoform NM OO3856 interleukin 1 receptor-like 1 isoform 2 NM 144701 interleukin 23 receptor precursor NM 004843 class I cytokine receptor NM 173065 interleukin 28 receptor, alpha isoform 3 NM OOO565 interleukin 6 receptor isoform 1 precursor NM 000584 interleukin 8 precursor LDR1 NM 175924 immunoglobulin-like domain containing receptor LKAP NM 176799 integrin-linked kinase-associated protein MPAD1 NM 017813 myo-inositol monophosphatase A3 NHBA NM 002192 inhibin beta. A precursor NHBE NM 031479 activin beta B NOC1 NM 017553 INO80 complex homolog 1 NPP5E NM 005540 inositol polyphosphate-5-phosphatase, 75 kDa NPPSF NM O14937 inositol polyphosphate-5-phosphatase F isoform US 2009/0163434 A1 Jun. 25, 2009 31

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description NTS5 NM 030628 integrator complex subunit 5 NTS7 NM O15434 integrator complex subunit 7 PO8 NM OO6390 importin 8 PP NM OO5897 intracisternal Aparticle-promoted polypeptide PPK NM O22755 inositol 1,3,4,5,6-pentakisphosphate 2-kinase QCC NM 018134 IQ motif containing C QSEC1 NM O14869 IQ motif and Sec7 domain 1 QSEC2 NM O15075 IQ motif and Sec7 domain 2 NM 001025242 interleukin-1 receptor-associated kinase 1 NM 016123 interleukin-1 receptor-associated kinase 4 NM 002198 interferon regulatory factor 1 NM 173576 hypothetical protein LOC283078 SG2OL1 NM O22767 interferon stimulated exonuclease gene SGF3G NM 006084 interferon-stimulated transcription factor 3, TCH NM 031483 itchy homolog E3 ubiquitin protein ligase TFG NM 030790 T-cell immunomodulatory protein TGA10 NM 003637 integrin, alpha 10 precursor TGA4 NM OOO885 integrin alpha 4 precursor TGAL NM OO2209 integrin alpha L precursor NM 002211 integrin beta 1 isoform 1 Aprecursor NM 002214 integrin, beta 8 NM OO4791 integrin, beta-like 1 (with EGF-like repeat NM 001001851 inter-alpha trypsin inhibitor heavy chain NM 198510 hypothetical protein LOC347365 NM O14216 inositol 1,3,4-triphosphate 5/6 kinase NM OO2221 D-myo-inositol-trisphosphate 3-kinase B NM147152 intersectin 2 isoform 2 NM OO6469 influenza virus NS1A binding protein isoform a NM 017592 intersex-like NM 175061 juxtaposed with another Zinc finger gene 1 NM O14876 hypothetical protein LOC9929 NM 003772 jerky homolog-like NM 032876 jub, ajuba homolog isoform 1 KAL1 NM 000216 Kallmann syndrome 1 protein KATNAL1 NM 001014380 katanin p60 subunit A-like 1 KBTBD6 NM 152903 kelch repeat and BTB (POZ) domain-containing 6 KBTBD8 NM 032505 T-cell activation kelch repeat protein KCNA7 NM 031886 potassium voltage-gate channel, shaker-related KCNB1 NM OO4975 potassium voltage-gate channel, Shab-related KCNHS NM 1393.18 potassium voltage-gate channel, Subfamily H, KCNH6 NM 030779 potassium voltage-gate channel, Subfamily H, KCNH8 NM 144633 potassium voltage-gate channel, Subfamily H, KCNJ 10 NM 002241 potassium inwardly-rec ifying channel, Subfamily KCN16 NM 018658 potassium inwardly-rec ifying channel J16 KCNJ8 NM 004982 potassium inwardly-rec ifying channel J8 KCNJ9 NM OO4983 potassium inwardly-rec ifying channel Subfamily KCNK1 NM OO2.245 potassium channel, Sub amily K, member 1 KCNK2 NM 001017424 potassium channel, Sub amily K, member 2 isoform KCNK3 NM 002246 potassium channel, Sub amily K, member 3 KCNK6 NM 004823 potassium channel, Sub amily K, member 6 KCNMA1 NM 001014797 arge conductance calcium-activated potassium KCNQ2 NM OO4518 potassium voltage-gate channel KQT-like protein KCNRG NM 1994.64 potassium channel regu ator isoform 2 KCNT2 NM 1985 03 potassium channel, Sub amily T, member 2 KCTD18 NM 152387 potassium channel tetramerisation domain KDELC2 NM 153705 KDEL (Lys-Asp-Glu-Leu) containing 2 KEAP1 NM 012289 kelch-like ECH-associa ed protein 1 KIAAO082 NM O15050 hypothetical protein LOC23070 KIAAO143 NM O15137 hypothetical protein LOC23167 KIAAO157 NM 032182 hypothetical protein LOC23172 KIAAO240 NM O15349 hypothetical protein LOC23506 KIAAO247 NM 014734 hypothetical protein LOC9766 KIAAO319 NM O14809 KIAAO319 KIAAO319L. NM O24874 polycystic kidney disease 1-like isoform a KIAAO355 NM 014686 hypothetical protein LOC9710 KIAAO367 NM O15225 BNIP2 motif containing molecule at the carboxyl KIAAO404 NM O15104 hypothetical protein LOC23130 KIAAO427 NM 014772 hypothetical protein LOC9811 KIAAO446 NM 014655 hypothetical protein LOC9673 KIAAO467 NM O15284 KIAAO467 protein US 2009/0163434 A1 Jun. 25, 2009 32

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq Transcript ID (Pruitt et al., 2005) Description NM 014774 hypothetical protein LOC9813 NM 207306 KIAAO495 NM 014732 hypothetical protein LOC9764 NM 014704 glycine-, glutamate-, NM O15058 hypothetical protein LOC23078 isoform a NM O14811 A6/DRIM (down-regulated in metastasis) NM O15229 hypothetical protein LOC23277 NM OO1031690 hypothetical protein LOC9715 NM O15328 KIAAO828 protein NM O14924 hypothetical protein LOC22863 NM 152257 hypothetical protein LOC25781 NM O15329 hypothetical protein LOC23383 NM O15314 hypothetical protein LOC23366 NM O15196 KIAAO922 protein NM O25176 hypothetical protein LOC22981 NM O25164 KIAA0999 protein NM 018999 granule cell antiserum positive 14 NM 020701 hypothetical protein LOC57461 NM 020444 hypothetical protein LOC57179 NM 017550 hypothetical protein LOC54531 NM 018689 KIAA11.99 NM 020717 hypothetical protein LOC57477 NM O2O775 hypothetical protein LOC57535 NM 020802 hypothetical protein LOC57562 NM O19593 hypothetical protein LOC56261 NM 020844 hypothetical protein LOC57604 NM020888 hypothetical protein LOC57648 NM 020894 hypothetical protein LOC57654 NM 020917 Zinc finger protein 14-like NM 018330 hypothetical protein LOC57698 NM 020940 hypothetical protein LOC57700 NM 020947 hypothetical protein LOC57707 NM 020954 hypothetical protein LOC57714 NM 020961 hypothetical protein LOC57721 NM 020970 hypothetical protein LOC57730 NM 030636 hypothetical protein LOC80820 NM 03.3393 hypothetical protein LOC85462 NM 032424 KIAA1826 protein NM 052909 hypothetical protein LOC153478 NM OO4523 kinesin family member 11 NM O15254 kinesin family member 13B NM O14875 kinesin family member 14 NM 004321 axonal transport of synaptic vesicles NM O15074 kinesin family member 1B isoform b NM 004.856 kinesin family member 23 isoform 2 NM OO4798 kinesin family member 3B NM OO2254 kinesin family member 3C NM 004984 kinesin family member 5A NM 022342 kinesin family member 9 isoform 1 NM 018240 kin of IRRE like NM 000222 v-kit Hardy-Zuckerman 4 feline sarcoma viral C3 NM 145275 kinesin light chain 2-like isoform b NM OO1032282 Kruppel-like factor 10 isoform b NM 003597 Kruppel-like factor 11 NM OO7249 Kruppel-like factor 12 isoform a NM O15995 Kruppel-like factor 13 NM 173484 Zinc finger protein 393 NM OO12O6 Kruppel-like factor 9 NM 020782 kelch domain containing 5 NM 207335 hypothetical protein LOC166348 NM 021633 kelch-like 12 NM OO7246 kelch-like 2, Mayven NM O14458 kelch-like 20 NM O14851 kelch-like 21 NM 032775 kelch-like NM 144711 kelch-like 23 NM O17415 kelch-like 3 (Drosophila) NM 020803 kelch-like 8 5 NM 012427 kallikrein 5 preproprotein 7 NM 005046 stratum corneum chymotryptic enzyme US 2009/0163434 A1 Jun. 25, 2009 33

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description KLRK1 NM OO7360 NKG2-D type II integral membrane protein KMO NM O03679 kynurenine 3-monooxygenase KPNA2 NM 002266 karyopherin alpha 2 KPNA3 NM OO2267 karyopherin alpha 3 KPNA4 NM O02268 karyopherin alpha 4 KRIT1 NM 001013406 krew interaction trapped 1 isoform 2 KRT10 NM 000421 keratin 10 KRT23 NM O15515 keratin 23 KRT2A NM 000423 keratin 2a KRT2B NM O15848 cytokeratin 2 KRT6IRS NM 033448 keratin 6 irs KRTAP10-4 NM 1986.87 keratin associated protein 10-4 KRTHB1 NM OO2281 keratin, hair, basic, 1 KRTHBS NM OO2.283 keratin, hair, basic, 5 KTI12 NM 138417 KTI12 homolog, chromatin associated L2HGDH NM 024.884 hypothetical protein LOC79944 L3MBTL2 NM 001003689 (3)mbt-like 2 isoform b L3MBTL3 NM OO1007102 (3)mbt-like 3 isoform b L3MBTL4 NM 173464 hypothetical protein LOC91133 LACE1 NM 145315 actation elevated LALBA NM OO2289 actalbumin, alpha-precursor LAMA3 NM 000227 aminin alpha 3 Su bunit isoform 2 LAMC1 NM 002293 aminin, gamma 1 precursor LAMC2 8891 aminin, gamma 2 isoform b precursor LAMP1 NM 005561 ysosomal-associa ed membrane protein 1 LAMP2 NM O 3995 ysosomal-associa ed membrane protein 2 LAMP3 NMO 4398 ysosomal-associa ed membrane protein 3 LAPTM4A NM O 4713 ysosomal-associa ed protein transmembrane 4 LARP2 NM 018078 La ribonucleoprotein domain family member 2 LARPS NM O15155 La ribonucleoprotein domain family, member 5 LASP1 NM 006148 LIM and SH3 protein 1 LASS2 NM 013384 LAG1 longevity assurance homolog 2 isoform 2 LASS3 NM 178842 hypothetical protein LOC204219 LASS6 NM 203463 ongevity assurance homolog 6 LAX1 NM O17773 ymphocyte transmembrane adaptor 1 LBH NM 030915 hypothetical protein DKFZp566J091 LDLR NM 000527 ow density lipoprotein receptor precursor LDLRAD3 NM 174902 hypothetical protein LOC143458 LDLRAP1 NM O15627 ow density lipoprotein receptor adaptor protein LDOC1L, NM 032287 hypothetical protein LOC84247 LEP NM OOO230 eptin precursor LEPREL1 NM 018192 eprecan-like 1 LEPROT NM 017526 eptin receptor gene-related protein LEPROTL1 NM O15344 eptin receptor overlapping transcript-like 1 LETM1 NM 012318 eucine Zipper-EF-hand containing transmembrane LGALS8 NM OO6499 galectin 8 isoform a LHFP NM OO5780 ipoma HMGIC fusion partner LHFPL2 NM 005779 ipoma HMGIC fusion partner-like 2 LHFPL3 NM 199000 ipoma HMGIC fusion partner-like 3 LHX6 NM O14368 LIM homeobox protein 6 isoform 1 LHX8 NM 001001933 LIM homeobox. 8 LIAS NM OO6859 ipoic acid synthetase isoform 1 precursor LIF NM 002309 eukemia inhibitory factor (cholinergic LILRAS NM 181986 eukocyte immunoglobulin-like receptor Subfamily LILRB1 NM OO6669 eukocyte immunoglobulin-like receptor, LIMA1 NM 016357 epithelial protein lost in neoplasm beta LIMK1 NM 002314 LIM domain kinase 1 isoform 1 LIN10 NM O251.87 in-10 LIN28 NM O24674 in-28 homolog LINAB NM 0221 65 in-7 homolog B LINS1 NM 181740 ines homolog 1 isoform 3 LIPEH NM 139248 ipase, member H precursor LITAF NM 004862 LPS-induced TNF-alpha factor LMO2 NM OO5574 LIM domain only 2 LMO3 NM 001001395 LIM domain only 3 LMOD3 NM 1982.71 eiomodin 3 (fetal) LNK NM 005475 ymphocyte adaptor protein LOC116143 NM 138458 Monad LOC116236 NM 198147 hypothetical protein LOC116236 LOC123688 NM 001013619 hypothetical protein LOC123688 US 2009/0163434 A1 Jun. 25, 2009 34

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description LOC124751 NM 213597 hypothetical protein LOC124751 LOC126248 NM 173479 hypothetical protein LOC126248 LOC128439 NM 139016 hypothetical protein LOC128439 LOC12928S NM 152994 Smooth muscle myosin heavy chain 11 isoform LOC130951 NM 138804 hypothetical protein LOC130951 LOC133619 NM 1308.09 hypothetical protein LOC133619 LOC134147 NM 1388.09 hypothetical protein LOC134147 LOC136263 NM 145268 hypothetical protein LOC136263 LOC148137 NM 144692 hypothetical protein LOC148137 LOC14962O hypothetical protein LOC149620 LOC151194 hypothetical protein LOC151194 LOC153222 hypothetical protein LOC153222 LOC153561 hypothetical protein LOC153561 LOC1581 60 7-beta-hydroxysteroid dehydrogenase type LOC162427 hypothetical protein LOC162427 LOC1688SO hypothetical protein LOC168850 LOC2O1895 hypothetical protein LOC201895 LOC2O3427 mitochondrial Solute carrier protein LOC2O3S47 hypothetical protein LOC203547 LOC22O594 TL132 protein LOC221091 hypothetical protein LOC221091 LOC222171 hypothetical protein LOC222171 LOC223075 hypothetical protein LOC223075 LOC283537 hypothetical protein LOC283537 LOC283SS1 NM 001012706 hypothetical protein LOC283551 LOC283849 NM 178516 hypothetical protein LOC283849 LOC284434 NM OO1.0075.25 hypothetical protein LOC284434 LOC284.912 NM 203375 hypothetical protein LOC284.912 LOC285382 NM 001025266 hypothetical protein LOC285382 LOC28S636 NM 175921 hypothetical protein LOC285636 LOC338328 NM 178172 high density lipoprotein-binding protein LOC339.745 NM 001001664 hypothetical protein LOC339745 LOC340843 NM 001013629 hypothetical protein LOC340843 LOC347273 NM 001018116 hypothetical protein LOC347273 LOC348262 NM 207368 hypothetical protein LOC348262 LOC387758 NM 203371 hypothetical protein LOC387758 LOC387790 NM 001013634 hypothetical protein LOC387790 LOC387873 NM 001013636 hypothetical protein LOC387873 LOC387882 NM 207376 hypothetical protein LOC387882 LOC387921 NM 001012754 hypothetical protein LOC387921 isoform a LOC38833S NM OO10043.13 hypothetical protein LOC388335 LOC38861O NM 001013642 hypothetical protein LOC388610 LOC38.8969 NM 001013649 hypothetical protein LOC38.8969 LOC389432 NM OO 103OO60 hypothetical protein LOC389432 LOC389634 NM 001012988 hypothetical protein LOC389634 LOC389936 NM 001013656 hypothetical protein LOC389936 LOC390980 NM 001023563 similar to Zinc finger protein 264 LOC3997O6 NM 001010910 hypothetical protein LOC3997O6 LOC400464 NM 001013670 hypothetical protein LOC400464 LOC4OO499 NM 001013671 hypothetical protein LOC400499 137 NM 214711 hypothetical protein LOC401 137 152 NM 001001701 hypothetical protein LOC401 152 410 NM OO1008742 hypothetical protein LOC401410 431 NM OO10O8745 hypothetical protein LOC4.01431 507 NM 00101.2278 hypothetical protein LOC4O1507 589 NM 001013687 hypothetical protein LOC4O1589 62O NM 001013688 hypothetical protein LOC4O1620 LOC4O2176 NM 00101.1538 hypothetical protein LOC402176 LOC440248 NM 199045 hypothetical protein LOC440248 LOC44O742 NM 001013710 hypothetical protein LOC440742 LOC44 136 NM 001013719 hypothetical protein LOC441136 LOC44 208 NM 001013723 hypothetical protein LOC441208 LOC44 268 NM 001013725 hypothetical protein LOC441268 LOC44 376 NM 001025357 hypothetical protein LOC441376 LOC442578 NM 001013739 hypothetical protein LOC442578 LOC493.829 NM OO10O8274 hypothetical protein LOC493829 LOCS1 49 NM 001017987 hypothetical protein LOC51149 isoform 2 LOCS1333 NM 016643 mesenchymal stem cell protein DSC43 LOCS1334 NM 016644 mesenchymal stem cell protein DSC54 LOCSS4251 NM 001024680 hypo le ica Ocil LOCSS42S1 US 2009/0163434 A1 Jun. 25, 2009 35

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description LOCS7149 NM 020424 hypothetical protein LOC57149 LOC6132O6 NM OO 1033016 myeloproliferative disease associated tumor LOC613266 NM OO1033516 hypothetical protein LOC613266 LOC619208 NM OO1033564 hypothetical protein LOC619208 LOC63928 NM O22097 hepatocellular carcinoma antigen gene 520 LOC63929 NM O22098 hypothetical protein LOC63929 LOC81558 NM O3O802 C/EBP-induced protein LOC90321 NM 00101.0851 hypothetical protein LOC90321 LOC90624 NM 181705 hypothetical protein LOC90624 LOC90639 NM OO1031617 hypothetical protein LOC90639 LOC94431 NM 145237 hypothetical protein LOC94431 LONPL NM 031490 peroxisomal LON protease-like LPGAT1 NM O14873 ySophosphatidylglycerol acyltransferase 1 LPHN3 NM O15236 atrophilin 3 precursor LPIN1 NM 145693 ipin 1 LPIN3 NM 022896 ipin 3 RA NM O22350 eukocyte-derived arginine aminopeptidase RA NM 004744 ecithin retinol acyltransferase FNS NM 152447 eucine rich repeat and fibronectin type III NM 052972 eucine-rich alpha-2-glycoprotein 1 NM O15541 eucine-rich repeats and immunoglobulin-like NM 002332 ow density lipoprotein-related protein 1 NM 013437 Suppression of tumorigenicity NM 018557 ow density lipoprotein-related protein 1B NM 018409 LRP2 binding protein NM 002334 ow density lipoprotein receptor-related protein NM 201550 eucine rich repeat containing 10 NM 130830 eucine rich repeat containing 15 NM O24512 eucine rich repeat containing 2 NM 018205 eucine rich repeat containing 20 isoform 3 NM 030626 eucine rich repeat containing 27 NM OO5512 eucine rich repeat containing 32 precursor NM 145258 eucine rich repeat containing 44 NM 144999 eucine rich repeat containing 45 NM O15516 Tsukushi NM 001005210 hypothetical protein LOC219527 NM 153260 hypothetical protein LOC255.252 NM 019594 eucine-rich repeat-containing 8 NM O15350 T-cell activation leucine repeat-rich protein NM 024548 eucine-rich repeats and IQ motif containing 2 NM 0328.08 eucine-rich repeat neuronal 6A NM 173491 LSM11, U7 small nuclear RNA associated NM 152344 hypothetical protein LOC124801 NM 181657 eukotriene B4 receptor NM 000428 atent transforming growth factor beta binding NM 002342 ymphotoxin beta receptor NM 032860 hypothetical protein LOC84946 NM 033631 eucine Zipper protein 1 NM 002349 ymphocyte antigen 75 NM OO10O2257 ySocardiolipin acyltransferase isoform 2 NM 000081 ysosomal trafficking regulator isoform 1 NM 002355 cation-dependent mannose-6-phosphate receptor NM OO5817 mannose 6 phosphate receptor binding protein 1 MA F1 NM 032272 MAF1 protein MA FF NM 012323 transcription factor MAFF MAGI2 NM 012301 membrane associated guanylate kinase, WW and PDZ MA NM OO5906 male germ cell-associated kinase MA NM 052886 mal, T-cell differentiation protein 2 MAN1A2 NM OO6699 mannosidase, alpha, class 1A, member 2 MAN2A2 NM 006122 mannosidase, alpha, class 2A, member 2 MANEAL NM 152496 hypothetical protein LOC149175 isoform 2 MA NM 02281.8 microtubule-associated proteins 1A1B light MA NM 002419 mitogen-activated protein kinase kinase kinase MA NM OO6301 mitogen-activated protein kinase kinase kinase MA NM OO3954 mitogen-activated protein kinase kinase kinase MA NM OO6609 mitogen-activated protein kinase kinase kinase MA NM OO2401 mitogen-activated protein kinase kinase kinase 3 MA NM OO5923 mitogen-activated protein kinase kinase kinase MA NM OO3188 mitogen-activated protein kinase kinase kinase 7 MA NM O05204 mitogen-activated protein kinase kinase kinase US 2009/0163434 A1 Jun. 25, 2009 36

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description MAP3K9 NM 033141 mitogen-activated protein kinase kinase kinase MAP6 NM 207577 microtubule-associated protein 6 isoform 2 MAP7 NM OO3980 microtubule-associated protein 7 MAPK NM OO2745 mitogen-activated protein kinase 1 MAPK4 NM OO2747 mitogen-activated protein kinase 4 MAPK9 NM OO2752 mitogen-activated protein kinase 9 isoform 1 MAPKBP1 NM 014994 mitogen-activated protein kinase binding protein MAPRE NM 012325 microtubule-associated protein, RP/EB family, MAPRE NM 012326 microtubule-associated protein, RP/EB family, MARCH2 NM 001005415 membrane-associated ring finger (C3HC4)2 MARCHS NM 017824 ring finger protein 153 MARCH6 NM OO5885 membrane-associated ring finger (C3HC4) 6 MARCH7 NM 022826 Axotrophin MARCH8 NM OO10O2265 cellular modulator of immune recognition MARK1 NM 018650 MAP microtubule affinity-regulating kinase 1 MARK4 NM 031417 MAP microtubule affinity-regulating kinase 4 MARS NM OO4990 methionine-tRNA synthetase MARVELD3 NM 001017967 MARVEL domain containing 3 isoform 1 MASTL NM 032844 microtubule associated serine/threonine MAT2B NM 013283 methionine adenosyltransferase II, beta isoform MAWBP NM 022129 MAWD binding protein isoform a MBDS NM 018328 methyl-CpG binding domain protein 5 MBNL NM 021038 muscleblind-like 1 isoform a MBNL3 NM 018388 muscleblind-like 3 isoform G MBP NM 001025 100 Golli-mbp isoform 2 MBTD NM O17643 mbt domain containing 1 MBTPS1 NM 003791 membrane-bound transcription factor site-1 MCAM NM OO6500 melanoma cell adhesion molecule MCF2L2 NM O15078 Rho family guanine-nucleotide exchange factor MCFD2 NM 139279 multiple coagulation factor deficiency 2 MCL NM O21960 myeloid cell leukemia sequence 1 isoform 1 MCM3 NM 002388 minichromosome maintenance protein 3 MCM4 NM 005914 minichromosome maintenance protein 4 MCMDC1 NM 153255 minichromosome maintenance protein domain MCOLN2 NM 153259 mucolipin 2 MDFIC NM 1990.72 MyoD family inhibitor domain containing isoform MDM4 NM 002393 mouse double minute 4 homolog MECP2 NM OO4992 methyl CpG binding protein 2 MECR NM 001024732 nuclear receptor-binding factor 1 isoform b MED12L NM 053002 hypothetical protein LOC116931 MED18 NM O17638 mediator of RNA polymerase II transcription, MED6 NM OO5466 mediator of RNA polymerase II transcription, METAP1 NM O15143 methionyl aminopeptidase 1 METTSD1 NM 152636 methyltransferase 5 domain containing 1 METTL2A NM 001005372 hypothetical protein LOC339175 METTL4 NM 022840 methyltransferase like 4 MFAP3L NM 001009554 microfibrillar-associated protein 3-like isoform MFAPS NM OO3480 microfibrillar associated protein 5 MFN2 NM O14874 mitofusin 2 MFSD4 NM 181644 hypothetical protein DKFZp761N1114 MGC11266 hypothetical protein LOC79172 MGC11332 hypothetical protein LOC84804 MGC13017 hypothetical protein LOC91368 MGC15476 hymus expressed gene 3-like MGC15619 hypothetical protein LOC84329 MGC16291 hypothetical protein LOC84856 MGC16385 hypothetical protein LOC928.06 MGC16703 hypothetical protein LOC113691 MGC19604 hypothetical protein LOC112812 isoform 1 MGC22001 hypothetical protein LOC197196 MGC24039 hypothetical protein LOC160518 MGC26718 hypothetical protein LOC440482 MGC26733 hypothetical protein LOC200403 MGC26816 hypothetical protein LOC164684 MGC2752 hypothetical protein LOC65996 MGC298.91 GA repeat binding protein, beta 2 MGC3123 hypothetical protein LOC79089 isoform 1 MGC32O20 hypothetical protein LOC91442 MGC32O7 hypothetical protein LOC84245 isoform 1 MGC34646 hypothetical protein LOC157807 US 2009/0163434 A1 Jun. 25, 2009 37

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description MGC34821 NM 173586 hypothetical protein LOC283238 MGC3SO48 NM 153208 hypothetical protein LOC124152 MGC3S440 NM 153220 hypothetical protein LOC147990 MGC39518 NM 173822 hypothetical protein LOC285172 MGC40069 NM 182615 hypothetical protein LOC348.035 MGC40405 NM 152789 hypothetical protein LOC257415 MGC42090 hypothetical protein LOC256130 MGC4268 hypothetical protein LOC83607 MGC4S438 hypothetical protein LOC146556 MGC4562 hypothetical protein LOC115752 MGC46SS hypothetical protein LOC84752 MGC48628 hypothetical protein LOC401145 MGC50372 hypothetical protein LOC253143 MGC52057 hypothetical protein LOC130574 MGCS2110 hypothetical protein LOC493.753 MGCS2498 hypothetical protein LOC348378 MGC70857 hypothetical protein LOC414919 MGC87631 hypothetical protein LOC339184 MGC9712 NM 152689 hypothetical protein LOC202915 MGEAS NM 012215 meningioma expressed antigen 5 (hyaluronidase) MGLL NM 001003794 monoglyceride lipase isoform 2 CA NM OOO247 MHC class I chain-related gene A protein CB NM 005931 MHC class I polypeptide-related sequence B NM 1774O1 Midnolin NM 001024937 misshapen? NIK-related kinase isoform 4 NM 002417 antigen identified by monoclonal antibody Ki-67 NMO14048 megakaryoblastic leukemia 2 protein NM 013255 muskelin 1, intracellular mediator containing NM 017572 MAP kinase-interacting serine/threonine kinase 2 NM 013446 makorin, ring finger protein, 1 NM O15166 megalencephalic leukoencephalopathy with NM 021230 myeloid lymphoi or mixed-lineage leukemia 3 NM 014727 myeloid lymphoi or mixed-lineage leukemia 4 NM 001009569 myeloid lymphoi or mixed-lineage leukemia LLT11 NM OO6818 MLLT11 protein LLT6 NM 005937 myeloid lymphoi or mixed-lineage leukemia LR1 NM 153686 transcription factor MLR1 MMACHC NM O15506 hypothetical protein LOC25974 MME NM OOO902 membrane metallo-endopeptidase MMP19 NM OO 1032360 matrix metallopro einase 19 isoform 2 precursor MMP2 NM 004530 matrix metallopro einase 2 preproprotein MMP23A NM 004659 matrix metallopro einase 23A precursor MMP23B NM OO6983 matrix metallopro einase 23B precursor MMP24 NM OO6690 matrix metallopro einase 24 preproprotein MMP3 NM 002422 matrix metallopro einase 3 preproprotein MMRN2 NM O24756 multimerin 2 MOBKL1A NM 173468 MOB1, Mps One Binder kinase activator-like 1A MOCS1 NM OO5943 molybdenum cofactor synthesis-step 1 protein MOG NM OO10O8228 myelin oligodendrocyte glycoprotein isoform MOGAT3 NM 178176 monoacylglycerol O-acyltransferase 3 MORF4L1 NM OO6791 MORF-related gene 15 isoform 1 MORF4L2 NM 012286 MORF-related geneX C PE1 NM 023075 metallophosphoesterase 1 isoform a precursor NM 000530 myelin protein zero NM 012219 muscle RAS oncogene homolog RC NM OO6471 myosin regulatory light chain MRCL3 NM OO5590 meiotic recombination 11 homolog A isoform 2 i NM 054031 G protein-coupled receptor MRGX3 6 NM 024026 mitochondrial ribosomal protein 63 NM 022061 mitochondrial ribosomal protein L17 NM 024540 mitochondrial ribosomal protein L24 NM 145212 mitochondrial ribosomal protein L30 NM 032112 mitochondrial ribosomal protein L43 isoform a NM 020409 mitochondrial ribosomal protein L47 isoform a o NM 004927 mitochondrial ribosomal protein L49 5 2 NM 178336 mitochondrial ribosomal protein L52 isoform a PS10 NM 018141 mitochondrial ribosomal protein S10 PS16 NM 016065 mitochondrial ribosomal protein S16 PS18B NM 014046 mitochondrial ribosomal protein S18B PS25 NM O22497 mitochondrial ribosomal protein S25 US 2009/0163434 A1 Jun. 25, 2009 38

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description MRPS36 NM 033281 mitochondrial ribosomal protein S36 MRRF NM 138777 mitochondrial ribosome recycling factor isoform MS4A10 NM 206893 membrane-spanning 4-domains, subfamily A, member MS4A7 NM 021201 membrane-spanning 4-domains, subfamily A, member MSH3 NM OO2439 mutS homolog 3 MSL2L1 NM 018133 ring finger protein 184 MSR1 NM 138715 macrophage scavenger receptor 1 isoform type 1 MSRB3 NM OO1031679 methionine sulfoxide reductase B3 isoform 2 MST1SO NM 032947 putative Small membrane protein NID67 MSTO1 NM 018116 Misato MTAC2D1 NM 152332 membrane targeting (tandem) C2 domain containing MTCH2 NM O14342 mitochondrial carrier homolog 2 MTERFD2 NM 1825O1 MTERF domain containing 2 MTF1 NM OO5955 metal-regulatory transcription factor 1 MTFMT NM 139242 methionyl-tRNA formyltransferase, mitochondrial MTHFD1 L, NM 015440 methylenetetrahydrofolate dehydrogenase (NADP+ MTHFD2 NM OO6636 methylene tetrahydrofolate dehydrogenase 2 MTMR12 NM O19061 myotubularin related protein 12 MTMR3 NM 021090 myotubularin-related protein 3 isoform c MTMRA NM 004686 myotubularin related protein 7 MTMR9 NM O15458 myotubularin-related protein 9 MUC17 NM 001004430 mucin 17 MUCDHL NM O17717 mu-protocadherin isoform 2 MULK NM 018238 multiple substrate lipid kinase MUM1L1 NM 152423 melanoma associated antigen (mutated) 1-like 1 MUTED NM 201280 Muted MVK NMOOO431 mevalonate kinase MXD1 NM 002357 MAX dimerization protein 1 MXI1 NM OO10O8541 MAX interactor 1 isoform c MXRA7 NM OO10O8529 transmembrane anchor protein 1 isoform 2 MYADM NM 001020818 myeloid-associated differentiation marker MYCL1 NM OO1033081 l-myc-1 proto-oncogene isoform 1 MYCN NM OO5378 v-myc myelocytomatosis viral related oncogene, MYF5 NM OO5593 myogenic factor 5 MYF6 NM 002469 myogenic factor 6 (herculin) MYLIP NM 013262 myosin regulatory light chain interacting MYLK NM 005965 myosin light chain kinase isoform 6 MYNN NM 018657 Myoneurin MYO10 NM 012334 myosin X MYO18A NM O78471 myosin 18A isoform a MYO1C NM 033375 myosin IC MYO1D NM O15194 myosin ID MYO3B NM 138995 myosin IIIB MYOHD1 NM OO 1033579 myosin head domain containing 1 isoform 2 MYOM1 NM OO3803 myomesin 1 MYOZ2 NM O16599 myOZenin 2 MYOZ3 NM 133371 myOZenin 3 MYT1L. NM O15025 myelin transcription factor 1-like N4BP1 NM 153029 Nedd4 binding protein 1 N4BP2 NM 018177 Nedd4 binding protein 2 NAGK NM 017567 N-Acetylglucosamine kinase NANOS1 NM 001009553 nanos homolog 1 isoform 2 NAPB NM O22080 N-ethylmaleimide-sensitive factor attachment NAPE-PLD NM 1989.90 N-acyl-phosphatidylethanolamine-hydrolyzing NARG1L, NM 018527 NMDA receptor regulated 1-like protein isoform NARS NM OO4539 asparaginyl-tRNA synthetase NAT11 NM O24771 hypothetical protein LOC79829 NAT12 NM 001011713 hypothetical protein LOC122830 NAV2 NM 145117 neuron navigator 2 isoform 2 NBEA NM O15678 Neurobeachin NBEAL1 NM 198945 neurobeachin-like 1 NBL NM O05380 neuroblastoma, Suppression of tumorigenicity 1 NBPF4 NM 152488 hypothetical protein LOC148545 NBR NM O05899 neighbor of BRCA1 gene 1 NBR2 NM OO5821 hypothetical protein LOC10230 NCF2 NM 000433 neutrophil cytosolic factor 2 NCK2 NM OO1004720 NCK adaptor protein 2 isoform A NCKAP1L, NM 005337 hematopoietic protein 1 NCOA3 NM OO6534 nuclear receptor coactivator 3 isoform b NCOA7 NM 181782 nuclear receptor coactivator 7 US 2009/0163434 A1 Jun. 25, 2009 39

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Transcript ID (Pruitt et al., 2005) Description NM 004.829 natural cytotoxicity triggering receptor 1 NM 001025579 nudE nuclear distribution gene E homolog like 1 NM OO2487 Necdin NM 002490 NADH dehydrogenase (ubiquinone) 1 alpha NM OO4549 NADH dehydrogenase (ubiquinone) 1, Subcomplex NM OO1 OO1503 NADH-ubiquinone oxidoreductase flavoprotein 3 NM OO4543 Nebulin NM OO6393 nebullette sarcomeric isoform NM O15277 ubiquitin-protein ligase NEDD4-like NM 021076 neurofilament, heavy polypeptide 200 kDa : NM 178170 NIMA-related kinase 8 NM 033116 NIMA related kinase 9 NM 013349 SCIRP10-related protein NM OO2499 neogenin homolog 1 NM 018092 neuropilin- and tolloid-like protein 2 NM OO6161 neurogenin 1 NM 024019 neurogenin 2 NM 000268 neurofibromin 2 isoform 1 NM O15090 neurofascin precursor NM OO6599 nuclear factor of activated T-cells 5 isoform c NM 172387 nuclear factor of activated T-cells, cytosolic NM 032815 nuclear factor of activated T-cells, NM 173164 cytoplasmic nuclear factor of activated T-cells NM OO4554 cytoplasmic nuclear factor of activated T-cells NM OO6164 nuclear factor (erythroid-derived 2)-like 2 NM OO5595 nuclear factor IA NMOO1001716 nuclear factor of kappa light polypeptide gene NM 0134.32 I-kappa-B-related protein NM 147134 nuclear transcription factor, X-box binding 1 NM OO6166 nuclear transcription factorY., beta NM 019850 neuronal guanine nucleotide exchange factor NM OO5598 nescient helix loop helix 1 NM 198270 Nance-Horan syndrome protein NM 020921 ninein isoform 2 NM O16533 ninjurin 2 NM 024.946 hypothetical protein LOC80011 NM 016101 60S ribosome subunit biogenesis protein NIP7 NM 144599 non-imprinted in Prader-Willi/Angelman syndrome NM O2O345 kappa B-ras 1 NM 001001349 NFKB inhibitor interacting Ras-like 2 NM OO2509 NK2 transcription factor related, 2 NM OO6167 NK3 transcription factor related, locus 1 NM O16231 nemo like kinase NM O15938 NMD3 homolog NM OO5793 nucleoside diphosphate kinase type 6 NM O22787 nicotinamide nucleotide adenylyltransferase 1 NM 021079 N-myristoyltransferase 1 NM OO4808 glycylpeptide N-tetradecanoyltransferase 2 NM OO6056 neuromedin U receptor 1 NM O2O167 neuromedin U receptor 2 NM O24654 hypothetical protein LOC79707 NM 1384.00 nucleolar protein with MIF4G domain 1 NOS1AP NM 014697 nitric oxide synthase 1 (neuronal) adaptor NOTCH2NL NM 203458 Notch homolog 2 N-terminal like protein PAL2 NM O24759 NIPA-like domain containing 2 PAL3 NM 020448 NIPA-like domain containing 3 PAS2 NM OO2518 neuronal PAS domain protein 2 PAS3 NM 022123 neuronal PAS domain protein 3 isoform 1 PAT NM OO2519 nuclear protein, ataxia-telangiectasia locus PC1 NM 000271 Niemann-Pick disease, type C1 PEPL1 NM 024663 aminopeptidase-like 1 PHP1 NM 000272 nephrocystin isoform 1 PHP3 NM 153240 nephronophthisis 3 PHS1 NM 004646 Nephrin PL NM 030769 N-acetylneuraminate pyruvate lyase PLOC4 NM O17921 nuclear protein localization 4 PNT NM OO 1033047 Nephronectin PTX1 NM OO2522 neuronal pentraxin I precursor PTXR NM O14293 neuronal pentraxin receptor isoform 1 PYSR NM OO6174 neuropeptide Y receptor Y5 US 2009/0163434 A1 Jun. 25, 2009 40

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description NR2E1 NM OO3269 nuclear receptor Subfamily 2, group E, member 1 NR2E3 NM O14249 photoreceptor-specific nuclear receptor isoform NR3C1 NM OOO176 nuclear receptor Subfamily 3, group C, member 1 NR4A2 NM 00618.6 nuclear receptor Subfamily 4, group A, member 2 NR4A3 NM OO6981 nuclear receptor Subfamily 4, group A, member 3 NRBF2 NM 030759 nuclear receptor binding factor 2 NRBP1 NM 013392 nuclear receptor binding protein NRIP2 NM 031474 nuclear receptor interacting protein 2 NRP2 NM 018534 neuropilin 2 isoform 4 precursor NSUN4 NM 199044 NOL1/NOP2 Sun domain family 4 protein NTSC2 NM 012229 5'-nucleotidase, cytosolic II NTSDC3 NM O16575 hypothetical protein LOC51559 isoform 2 NTSE NM OO2526 5' nucleotidase, ecto NTN4 NM 021229 netrin 4 NTRK2 NM OO1007097 neurotrophic tyrosine kinase, receptor, type 2 NTSR1 NM OO2531 neurotensin receptor 1 NUAK1 NM O14840 AMPK-related protein kinase 5 NUBP1 NM 002484 nucleotide binding protein 1 (MiniD homolog, E. NUBPL NM O25152 nucleotide binding protein-like NUDT4 NM O19094 nudix-type motif 4 isoform alpha NUFIP2 NM O2O772 82-kD FMRP Interacting Protein NUP160 NM O15231 160 kDa NUP35 NM OO10O8544 kDa isoform b NUP43 NM 198887 kDa NUP62 NM 012346 kDa NUP98 NM 016320 nucleoporin 98 kD isoform 1 NUPL1 NMOO10O8564 nucleoporin like 1 isoform b NUSAP1 NM 016359 nucleolar and spindle associated protein 1 NY-SAR-48 NM 001011699 sarcoma antigen NY-SAR-48 isoform b OACT2 NM 138799 O-acyltransferase (membrane bound) domain OACTS NM OO5768 gene rich cluster, C3f gene OAS2 NM O16817 2'-5'-oligoadenylate synthetase 2 isoform 1 OATL1 NM OO1 OO6113 ornithine aminotransferase-like 1 isoform 1 OBFC2A NM OO1031716 hypothetical protein LOC64859 OBFC2B NM 024068 hypothetical protein LOC79035 OCLN NM OO2538 Occludin OCRL NM 000276 phosphatidylinositol polyphosphate 5-phosphatase OGDH NM OO10O3941 oxoglutarate (alpha-ketoglutarate) dehydrogenase OGG1 NM O16827 8-oxoguanine DNA glycosylase isoform 2c OGT NM OO3605 O-linked GlcNAc transferase isoform 3 OLIG1 NM 138983 oligodendrocyte transcription factor 1 OPA3 NM 001017989 OPA3 protein isoform a OPHN1 NM OO2547 oligophrenin 1 OPTN NM OO10O8211 Optineurin OR7D2 NM 175883 hypothetical protein LOC162998 ORC6L NM O14321 origin recognition complex subunit 6 ORMDL3 NM 13928O ORM1-like 3 OSBPL2 NM O14835 oxysterol-binding protein-like protein 2 isoform OSBPLS NM 020896 oxysterol-binding protein-like protein 5 isoform OSCAR NM 206817 Osteoclast-associated receptor isoform 2 OSM NM 020530 oncostatin M precursor OSR1 NM 145260 odd-skipped related 1 OSTM1 NM 014028 Osteopetrosis associated transmembrane protein OTUD4 NM 199324 OTU domain containing 4 protein isoform 1 OTUD6A NM 207320 HIN-6 protease OTX1 NM 014562 orthodenticle 1 OXR1 NM 181354 oxidation resistance 1 P2RX4 NM OO2560 purinergic receptor P2X4 isoform a P2RX7 NM OO2562 purinergic receptor P2X7 isoform a P2RY13 NM 023914 purinergic receptor P2Y, G-protein coupled, 13 P2RY14 NM O14879 purinergic receptor P2Y, G-protein coupled, 14 P2RY6 NM 004154 pyrimidinergic receptor P2Y6 P2RY8 NM 178129 G-protein coupled purinergic receptor P2Y8 P4HA3 NM 182904 prolyl 4-hydroxylase, alpha III subunit PABPC5 NM 080832 poly(A) binding protein, cytoplasmic 5 PACSIN1 NM 020804 protein kinase C and casein kinase Substrate in PADI1 NM 013358 peptidylarginine deiminase type I PAF1 NM O19088 Paf1, RNA polymerase II associated factor, PAFAH1B1 NM 000430 platelet-activating factor acetylhydrolase, PAFAH1B2 NM OO2572 platelet-activating factor acetylhydrolase, US 2009/0163434 A1 Jun. 25, 2009 41

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description PAFAH2 NM 000437 platelet-activating factor acetylhydrolase 2 PAG1 NM 018440 phosphoprotein associated with glycosphingolipid PAICS NM OO6452 phosphoribosylaminoimidazole carboxylase PAK2 NM OO2577 p21-activated kinase 2 PALLD NM 016081 Palladin PALM2-AKAP2 NM OO72O3 PALM2-AKAP2 protein isoform 1 PAM NM OOO919 peptidylglycine alpha-amidating monooxygenase PANK1 NM 138316 pantothenate kinase 1 isoform gamma PANK3 NM O24594 pantothenate kinase 3 PANX1 NM O15368 pannexin 1 PANX2 NM 052839 pannexin 2 PAPD1 NM 018109 PAP associated domain containing 1 PAPOLA NM 032632 poly(A) polymerase alpha PAPOLB NM 02014.4 poly(A) polymerase beta (testis specific) PAPOLG NM 022894 poly(A) polymerase gamma PAPPA NM OO2581 pregnancy-associated plasma protein A NM O17705 membrane progestin receptor gamma NM 032521 PAR-6 beta NM 032510 PAR-6 gamma protein NM OO2582 poly(A)-specific ribonuclease (deadenylation NM 017554 poly (ADP-ribose) polymerase family, member 14 NM 020213 poly (ADP-ribose) polymerase family, member 6 NM 018492 T-LAK cell-originated protein kinase NM 021635 prostate and breast cancer overexpressed 1 NM OO6195 pre-B-cell leukemia transcription factor 3 NM 020524 pre-B-cell leukemia transcription factor NMOO3884 p300, CBP-associated factor NM 032967 protocadherin 11 X-linked isoform b precursor NM 032971 protocadherin 11 Y-linked isoform a NM 022843 protocadherin 20 HA1 NM 018900 protocadherin alpha 1 isoform 1 precursor HA10 NM 018901 protocadherin alpha 10 isoform 1 precursor HA11 NM 018902 protocadherin alpha 11 isoform 1 precursor HA12 NM 018903 protocadherin alpha 12 isoform 1 precursor HA13 NM 018904 protocadherin alpha 13 isoform 1 precursor NM 018905 protocadherin alpha 2 isoform 1 precursor A3 NM 018906 protocadherin alpha 3 isoform 1 precursor A4 NM 018907 protocadherin alpha 4 isoform 1 precursor AS NM 018908 protocadherin alpha 5 isoform 1 precursor A6 NM 018909 protocadherin alpha 6 isoform 1 precursor A7 NM 018910 protocadherin alpha 7 isoform 1 precursor A8 NM 018911 protocadherin alpha 8 isoform 1 precursor A9 NM 031857 protocadherin alpha 9 isoform 1 precursor AC1 NM 018898 protocadherin alpha subfamily C, 1 isoform 1 HAC2 NM 018899 protocadherin alpha subfamily C, 2 isoform 1 HB9 NM 019119 protocadherin beta 9 precursor HGA7 NM 032087 protocadheringamma Subfamily A, 7 isoform 2 PCGF6 NM 001011663 polycomb group ring finger 6 isoform a PCK NM OO2591 cytosolic phosphoenolpyruvate carboxykinase 1 PCMTD1 NM 052937 hypothetical protein LOC115294 PCN C NM O2O357 PEST-containing nuclear protein PCNX NM O14982 pecanex homolog PCNXL2 NM 014801 pecanex-like 2 PCSK2 NM OO2594 proprotein convertase Subtilisinkexin type 2 PCSK6 NM 138323 paired basic cleaving system 4 PCYOX1 NM O16297 prenylcysteine oxidase 1 PCYT1B NM 004845 phosphate cytidylyltransferase 1, choline, beta PDAP1 NM O14891 PDGFA associated protein 1 PDCD1LG2 NM O25239 programmed cell death 1 ligand 2 PDCD4 NM O14456 programmed cell death 4 isoform 1 PDCD7 NM 005707 programmed cell death 7 PDDC1 NM 182612 hypothetical protein LOC347862 PDE1B NM 000924 phosphodiesterase 1B, calmodulin-dependent PDE3B NM OOO922 phosphodiesterase 3B, c0MP-inhibited PDE4A NM OO62O2 phosphodiesterase 4A, cAMP-specific PDE4B NM 002600 phosphodiesterase 4B, cAMP-specific isoform 1 PDE4C NM OOO923 phosphodiesterase 4C, cAMP-specific PDE4DIP NM OO10O2811 phosphodiesterase 4D interacting protein isoform PDE5A NM 001083 phosphodiesterase 5A isoform 1 PDE7A NM 002604 phosphodiesterase 7A isoform b US 2009/0163434 A1 Jun. 25, 2009 42

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description DE7B NM 018945 phosphodiesterase 7B DE8A NM 002605 phosphodiesterase 8A isoform 1 DGFB NM 002608 platelet-derived growth factor beta isoform 1, DGFC NM O16205 platelet-derived growth factor C precursor DGFD NM O25208 platelet derived growth factor D isoform 1 DGFRA NM OO62O6 platelet-derived growth factor receptor alpha DGFRB NM 002609 platelet-derived growth factor receptor beta HX NM OO3477 complex, component X K1L NM 152835 PDLIM1 interacting kinase 1 like K1 NM 002610 pyruvate dehydrogenase kinase, isozyme 1 K4 NM 002612 pyruvate dehydrogenase kinase, isoenzyme 4 LIM4 NM OO3687 PDZ and LIM domain 4 LIMS NM 00101.1513 PDZ and LIM domain 5 isoform b PK1 NM 002613 3-phosphoinositide dependent protein kinase-1 PN NM 001006624 ung type-I cell membrane-associated PR NM O17990 pyruvate dehydrogenase phosphatase regulatory RG1 NM O3O815 p53 and DNA damage-regulated protein ZD11 NM 016484 PDZ domain containing 11 NM 018441 peroxisomal trans-2-enoyl-CoA reductase NM OO6210 paternally expressed 3 NM 021255 pellino 2 NM O15946 pelota homolog NM 022817 period 2 isoform 1 NM 022121 PERP, TPS3 apoptosis effector NM O22574 PERQ amino acid rich, with GYF domain 1 NM 057174 peroxisomal biogenesis factor 16 isoform 2 NM 002857 peroxisomal biogenesis factor 19 NM O17929 peroxisome biogenesis factor 26 NM O16559 PXR2b protein NM OO2620 platelet factor 4 variant 1 NM OO6212 6-phosphofructo-2-kinase/fructose-2, NM 004566 6-phosphofructo-2-kinase/fructose-2, NM OO2627 phosphofructokinase, platelet NM OO2628 profilin 2 isoform b NM O14224 pepsinogen 5, group I (pepsinogen A) NM O24989 GPI deacylase NM 152595 piggyBac transposable element derived 4 NM O24554 piggyBac transposable element derived 5 NM O14485 prostaglandin-D synthase NM 002632 acental growth factor, vascular endothelial NM 173582 hosphoglucomutase 2-like 1 NM O21965 hosphoglucomutase 5 NM 023923 hosphatase and actin regulator 4 NM 002636 HD finger protein 1 isoform a NM O 6119 HD finger protein 11 NM O S288 HD finger protein 15 NM 024900 ade1 protein short isoform NM O05392 PHD finger protein 2 isoform a 6436 HD finger protein 20 NM O24297 HD finger protein 23 NM 001015877 HD finger protein 6 isoform 1 5107 HD finger protein 8 HKG1 NM OO6213 phosphorylase kinase, gamma 1 (muscle) HLDB3 NM 198850 pleckstrin homology-like domain, family B, HLPPL NM O SO20 PH domain and leucine rich repeat protein HTF2 NM 020432 putative homeodomain transcription factor 2 HYHIP NM O 4759 phytanoyl-CoA hydroxylase interacting protein NM O S886 protease inhibitor 15 preproprotein NM 005482 phosphatidylinositol glycan, class Kprecursor NM 145167 PIG-M mannosyltransferase NM 032634 phosphatidylinositol glycan, class O isoform 1 7861 GPI-mannosyltransferase subunit NM OO5026 phosphoinositide-3-kinase, catalytic, delta NM 181504 phosphoinositide-3-kinase, regulatory Subunit, NM 005O27 phosphoinositide-3-kinase, regulatory Subunit 2 5553 phosphoinositide-binding protein PIP3-E NM OO 1031687 hosphatidylinositol-4-phosphate 5-kinase, type NM O24779 hosphatidylinositol-4-phosphate 5-kinase, type NM OO10O2881 hosphatidylinositol-3- POX 6518 L-pipecolic acid oxidase US 2009/0163434 A1 Jun. 25, 2009 43

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Transcript ID (Pruitt et al., 2005) Description NM OO6224 phosphatidylinositol transfer protein, alpha NM 002653 paired-like homeodomain transcription factor 1 NM 018068 piwi-like 2 NM OOO296 polycystin 1 isoform 2 precursor NM OOO297 polycystin 2 NM OO6071 receptor for egg jelly-like protein precursor NM 138694 polyductin isoform 1 NM OO6823 cAMP-dependent protein kinase inhibitor alpha NM 007066 cAMP-dependent protein kinase inhibitor gamma NM OO42O3 protein kinase Myt1 isoform 1 NM 004571 PBX knotted 1 homeobox 1 isoform 1 NM OOO299 akophilin 1 isoform 1b NM 001004426 hospholipase A2, group VI isoform b NM 021796 acenta-specific 1 NM 153375 acenta-specific 2 NM 182832 acenta-specific 4 NM 002655 eiomorphic adenoma gene 1 NM 002657 eiomorphic adenoma gene-like 2 NM 002658 urokinase plasminogen activator preproprotein NM 001005376 asminogen activator, urokinase receptor NM 153021 hospholipase B1 B NM O15192 hosphoinositide-specific phospholipase C beta 1 NM 014996 hospholipase C-like 3 NM 001005473 hosphatidylinositol-specific phospholipase C, X NM 002662 hospholipase D1, phophatidylcholine-specific NM 012388 allidin NMOO1OO1974 eckstrinhomology domain containing, family A NM O19091 eckstrin homology domain containing, family A NM O14935 hosphoinositol 3-phosphate-binding protein-3 NM O17958 eckstrin homology domain containing, family B NM O24613 hafin 2 NM 0010298.84 eckstrin homology domain containing, family G NM 018173 eckstrin homology domain containing, family G NM 014798 eckstrin homology domain containing, family M NM O252O1 H domain-containing protein N NM 002666 erilipin NM 002670 plastin 1 NM O2O360 phospholipid scramblase 3 NM 020353 phospholipid scramblase 4 NM 020405 exin domain containing 1 precursor LXNA1 NM 032242 exin A LXNA4B NM 181775 hypothetical protein LOC91584 LXNC1 NM OO5761 exin C PMAIP1 NM 021127 horbol-12-myristate-13-acetate-induced protein PNKD NM O15488 myofibrillogenesis regulator 1 isoform 1 PNPLA1 NM 173676 patatin-like phospholipase domain containing 1 PNPLA4 NM 004650 GS2 gene PODN NM 153703 Podocan POFUT1 NM O15352 protein O-fucosyltransferase 1 isoform 1 POLDIP2 NM O15584 DNA polymerase delta interacting protein 2 POLH NM OO6502 polymerase (DNA directed), eta POLQ NM 199420 DNA polymerase theta POLR1E NM 022490 RNA polymerase I associated factor 53 POLR3A NM 007055 polymerase (RNA) III (DNA directed) polypeptide POLR3K NM 016310 DNA directed RNA polymerase III polypeptide K PON2 NM OOO305 paraoXonase 2 isoform 1 POU3F2 NM OO5604 POU domain, class 3, transcription factor 2 POU4F2 NM 004575 POU domain, class 4, transcription factor 2 POU6F1 NM OO2702 POU domain, class 6, transcription factor 1 PPAPDC3 NM 032728 phosphatidic acid phosphatase type 2 domain PPARA NM 001001928 peroxisome proliferative activated receptor, PPARD NM OO6238 peroxisome proliferative activated receptor, PPGB NM OOO3O8 protective protein for beta-galactosidase PPM1A NM 177951 protein phosphatase 1A isoform 2 PPM1B NM OO1033556 protein phosphatase 1B isoform 4 PPM1E NM O14906 protein phosphatase 1E PPM1K NM 152542 protein phosphatase 1 K (PP2C domain containing) PPP1R12B NM 002481 protein phosphatase 1, regulatory (inhibitor) PPP1R13B NM O15316 protein phosphatase 1, regulatory (inhibitor) PPP1R14C NM 030949 protein phosphatase 1, regulatory (inhibitor) US 2009/0163434 A1 Jun. 25, 2009 44

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description NM 032833 protein phosphatase 1, regulatory subunit 15B NM OO6741 protein phosphatase 1, regulatory (inhibitor) NM O24607 protein phosphatase 1, regulatory (inhibitor) NM OO5398 protein phosphatase 1, regulatory (inhibitor) NM 002715 protein phosphatase 2, catalytic Subunit, alpha NM 002716 beta isoform of regulatory subunit A, protein NM 002717 alpha isoform of regulatory subunit B55, protein NM 002718 protein phosphatase 2, regulatory subunit B", NM OOO944 protein phosphatase 3 (formerly 2B), catalytic NM OOO945 protein phosphatase 3, regulatory subunit B, NM 002721 protein phosphatase 6, catalytic Subunit PTC 7 NM 139283 T-cell activation protein phosphatase 2C QLC2 NM O17765 PQ loop repeat containing 2 RAP1 NM 1452O2 proline-rich acidic protein 1 DM10 NM 020228 PR domain containing 10 isoform 1 DM12 NM 021619 PR domain containing 12 DM14 NM 024504 PR domain containing 14 DM2 NM OO1007257 retinoblastoma protein-binding zinc finger EL NM 002725 proline arginine-rich end leucine-rich repeat EP NM OO6036 prolyl endopeptidase-like EX1 NM 020820 PREX1 protein NM 005042 proline-rich protein HaeIII subfamily 2 C285 NM 033405 PPAR-alpha interacting complex protein 285 CKLE2 NM 198859 prickle-like 2 KAB2 NM OO5399 AMP-activated protein kinase beta 2 KACB NM OO2731 cAMP-dependent protein kinase catalytic subunit KAR1A NMOO2734 cAMP-dependent protein kinase, regulatory KCH NM OO6255 protein kinase C, eta KD3 NM OO5813 protein kinase D3 NM 005044 protein kinase, X-linked NM 002760 protein kinase, Y-linked N D NM 012409 prion-like protein doppel preproprotein NM 000311 prion protein preproprotein NM 014117 hypothetical protein LOC29035 NM 018607 hypothetical protein LOC55471 isoform 2 NM 032414 prokineticin 1 NM 007198 proline synthetase co-transcribed homolog NM 004697 PRP4 pre-mRNA processing factor 4 homolog NM OO1031698 Huntingtin interacting protein C isoform 1 NM 003.913 serine/threonine-protein kinase PRP4K RR 1 NM 018304 hypothetical protein LOC55771 RRS NM 001017528 proline rich 5 (renal) isoform 2 NM OOO950 proline rich Gla (G-carboxyglutamic acid) 1 NM 024081 proline rich Gla (G-carboxyglutamic acid) 4 RRT 3 NM 207351 hypothetical protein LOC285368 RRX1 NM OO6902 paired mesoderm homeobox. 1 isoform pmX-1a RSS35 NM 153362 protease, serine, 35 PSCD1 NM 004762 pleckstrin homology, Sect and coiled coil PSD NM OO2779 pleckstrin and Sect domain containing PSD3 NM O15310 ADP-ribosylation factor guanine nucleotide PSG3 NM 021016 pregnancy specific beta-1-glycoprotein 3 PSMC3IP NM O16556 TBP-1 interacting protein isoform 2 PSMD12 NM 002816 proteasome 26S non-ATPase subunit 12 isoform 1 PSMD5 NM 005047 proteasome 26S non-ATPase subunit 5 PSTPIP2 NM 024430 proline-Serine-threonine phosphatase interacting PTAFR NM OOO952 platelet-activating factor receptor PTDS NM 014754 phosphatidylserine synthase 1 PTGDR NM OOO953 prostaglandin D2 receptor PTGER3 NM 198718 prostaglandin E receptor 3, Subtype EP3 isoform PTGER4 NM OOO958 prostaglandin E receptor 4, subtype EP4 NM 020440 prostaglandin F2 receptor negative regulator PTGIS NM OOO961 prostaglandin I2 (prostacyclin) synthase 1 NM OOO962 prostaglandin-endoperoxide synthase 1 isoform 1 PTHLH NM 198965 parathyroid hormone-like hormone isoform 1 NM OO5607 PTK2 protein tyrosine kinase 2 isoform b NM OO5975 PTK6 protein tyrosine kinase 6 NM 002822 twinfilin isoform 1 PTP4A1 NM OO3463 protein tyrosine phosphatase type IVA, member 1 PTPD NM 152422 protein tyrosine phosphatase domain containing 1 PTPN1 NM 002827 protein tyrosine phosphatase, non-receptor type US 2009/0163434 A1 Jun. 25, 2009 45

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description PTPN12 NM 002835 protein tyrosine phosphatase, non-receptor type PTPN3 NM 002829 protein tyrosine phosphatase, non-receptor type PTPN4 NM 002830 protein tyrosine phosphatase, non-receptor type PTPNS1 NM 080792 protein tyrosine phosphatase, non-receptor type PTPRO NM 002848 receptor-type protein tyrosine phosphatase O PTPRT NM 007050 protein tyrosine phosphatase, receptor type, T PTRF NM 012232 polymerase I and transcript release factor NM OO5859 purine-rich element binding protein A NM 033224 purine-rich element binding protein B NM O06505 poliovirus receptor NM 002856 poliovirus receptor-related 2 (herpesvirus entry NM 002862 brain glycogen phosphorylase NM 206853 quaking homolog, KH domain RNA binding isoform NM O14298 quinolinate phosphoribosyltransferase NM 018292 glutaminyl-tRNA synthase NM 001029875 R7 binding protein NM O16131 ras-related GTP-binding protein RAB10 NM 004663 Ras-related protein Rab-1 1A NM OO1002233 Rab coupling protein isoform 2 NM 032932 B11 family interacting protein 4 (class II) NM O15470 B11 family interacting protein 5 (class I) NM O14999 B21, member RAS oncogene family NM O2O673 S-related protein RAB-22A NM O16277 -related protein Rab-23 NM OO4580 -related protein Rab-27A NM 001017979 s B28, member RAS oncogene family isoform 1 NMO14488 B30, member RAS oncogene family NM OO6868 B31, member RAS oncogene family NM OO6861 B35, member RAS oncogene family NM OO1 OO6638 B37, member RAS oncogene family isoform 2 NM 002867 B3B, member RAS oncogene family NM 013401 B3A interacting protein (rabin3)-like 1 NM 198490 B43 protein NM 002868 B5B, member RAS oncogene family NM OO3929 B7, member RAS oncogene family-like 1 NM O16530 B8E3, member RAS oncogene family NM 004703 rabaptin, RAB GTPase binding effector protein 1 NM 012197 B GTPase activating protein 1 NM 013412 B, member of RAS oncogene family-like 2A NM OO10O3789 B, member of RAS oncogene family-like 2B NM O22777 B, member RAS oncogene family-like 5 CGA. P1 NM 013277 GTPase activating protein 1 NM 002853 D1 homolog isoform 1 NM 002873 D17 homolog isoform 1 NM O2O165 s replication repair protein hRAD18p NM 002874 excision repair protein RAD23 homolog B NM 002875 D51 homolog protein isoform 1 NM 002879 . D52 homolog isoform alpha G1 A. P1 NM 018845 stromal cell protein NM O22749 retinoic acid induced 16 NM 020338 retinoic acid induced 17 LBP1 NM OO6788 ralAbinding protein 1 LGPS1 NM 014636 Ral GEF with PH domain and SH3 binding motif 1 LGPS2 NM 018037 Ral GEF with PH domain and SH3 binding motif 2 NM OO6325 ras-related nuclear protein NM OO2271 RAN binding protein 5 NM 001010935 RAP1A, member of RAS oncogene family NM 002886 RAP2B, member of RAS oncogene family BLS NM 021183 RAP2C, member of RAS oncogene family NM 005312 guanine nucleotide-releasing factor 2 isoform a NM 007023 Rap guanine nucleotide exchange factor (GEF) 4 NM 016339 Rap guanine nucleotide exchange factor NM 213589 Ras association and pleckstrin homology domains O NM 020761 Raptor S. NM OOO965 retinoic acid receptor, beta isoform 1 NM 016084 RAS, dexamethasone-induced 1 RASGEF1A NM 145313 RasGEF domain family, member 1A NM O23940 RAS-like family 11 member B NM O16563 RAS-like, family 12 protein NM 014737 Ras association domain family 2 US 2009/0163434 A1 Jun. 25, 2009 46

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description RASSF6 NM 177532 Ras association (RalGDSAF-6) domain family 6 RASSF8 NM 007211 Ras association (RalGDSAF-6) domain family 8 RAVER2 NM 018211 ribonucleoprotein, PTB-binding 2 RB1 NM OOO321 retinoblastoma 1 RB1CC1 NM 014781 Rb1-inducible coiled coil protein 1 RBBPS NM 005057 retinoblastoma binding protein 5 RBBP7 NM 002893 retinoblastoma binding protein 7 RBJ NM O16544 Ras-associated protein Rap1 RBL1 NM 002895 retinoblastoma-like protein 1 isoform a RBL2 NM OO5611 retinoblastoma-like 2 (p130) RBM12 NM OO6047 RNA binding motif protein 12 RBM12B NM 203390 hypothetical protein LOC389677 RBM13 NM 032509 RNA binding motif protein 13 RBM1SB NM 013286 RNA binding motif protein 15B RBM7 NM 016090 RNA binding motif protein 7 RBPMS NM OO6867 RNA-binding protein with multiple splicing RCCD1 NM 001017919 hypothetical protein LOC91433 RCOR2 NM 173587 REST corepressor 2 RECQL5 NM 001003715 RecQ protein-like 5 isoform 2 REEP1 NM 022912 receptor expression enhancing protein 1 REEP3 NM OO1OO1330 receptor expression enhancing protein 3 REEP5 NM O05669 receptor accessory protein 5 REL NM OO2908 V-rel reticuloendotheliosis viral oncogene RERE NM 012102 atrophin-1 like protein RET NM 020975 ret proto-oncogene isoform a REXO1L1 NM 172239 exonuclease GOR RFC2 NM002914 replication factor C2 (40 kD) isoform 2 RFK NM 018339 riboflavin kinase RFX2 NM OOO635 regulatory factor X2 isoform a RFX5 NM 000449 regulatory factor X, 5 RFXAP NM 000538 regulatory factor X-associated protein RG9MTD3 NM 144964 RNA (guanine-9-) methyltransferase domain RGL1 NM O15149 ral guanine nucleotide dissociation RGMA NM 020211 RGM domain family, member A RGMB NM 001012761 RGM domain family, member B isoform 1 precursor RGPD5 NM 005054 RANBP2-like and GRIP domain containing 5 isoform RGS3 NM 021106 regulator of G-pro ein signalling 3 isoform 2 RGS4 NM OO5613 regulator of G-pro ein signaling 4 RGSS NM OO3617 regulator of G-pro ein signalling 5 RGS9BP NM 207391 RGS9 anchor protein RHBDL2 NM 017821 rhomboid-related protein 2 RHO NM 000539 Rhodopsin RHOA NM OO1664 ras homolog gene family, member A RHOBTB1 NM OO 1032380 Rho-related BTB domain containing 1 RHOBTB3 NM O14899 rho-related BTB domain containing 3 RHOC NM 175744 ras homolog gene family, member C RHOT1 NM OO1033566 ras homolog gene family, member T1 isoform 2 RHOV NM 133639 ras homolog gene family, member V RIC3 NM O24557 resistance to inhibitors of cholinesterase 3 RIMBP2 NM O15347 RIM-binding protein 2 RIMS4 NM 182970 regulating synaptic membrane exocytosis 4 RIOK3 NM OO3831 SudD Suppressor of bimD6 homolog isoform 1 RIPKS NM O15375 receptor interacting protein kinase 5 isoform 1 RKHD1 NM 203304 ring finger and KH domain containing 1 RKHD2 NM 016626 ring finger and KH domain containing 2 RNASEL NM 021133 ribonuclease L. RND3 NM OO51.68 ras homolog gene family, member E RNF11 NM O14372 ring finger protein 11 RNF12 NM 016120 ring finger protein 12 RNF125 NM O17831 ring finger protein 125 RNF128 NM O24539 ring finger protein 128 isoform 2 RNF141 NM 016422 ring finger protein 141 RNF144 NM 014746 ring finger protein 144 RNF149 NM 173647 ring finger protein 149 RNF157 NM 052916 ring finger protein 157 RNF170 NM 03.0954 ring finger protein 170 RNF18O NM 178532 ring finger protein 180 RNF19 NM O15435 ring finger protein 19 RNF2 NM 007212 ring finger protein 2 RNF24 NM 007219 ring finger protein 24 US 2009/0163434 A1 Jun. 25, 2009 47

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description RNF31 NM O17999 ring finger protein 31 RNF38 NM O22781 ring finger protein 38 isoform 1 RNF NM OO2938 ring finger protein 4 RNF6 NM OO5977 ring finger protein 6 isoform 1 RNF8 NM OO3958 ring finger protein 8 isoform 1 RNH1 NM OO2939 ribonucleasef angiogenin inhibitor RNMT NM 003799 RNA (guanine-7-) methyltransferase RNMTL1 NM 018146 RNA methyltransferase like 1 RNPC1 NM 183425 RNA-binding region containing protein 1 isoform RNPS1 NM OO6711 RNA-binding protein S1, serine-rich domain RNUXA NM 032177 RNA U. Small nuclear RNA export adaptor ROCK2 NM 004.850 Rho-associated, coiled-coil containing protein RA NM OO2943 RAR-related orphan receptor A isoform c s RC NM 001001523 RAR-related orphan receptor C isoform b PA2 NM 002.946 replication protein A2, 32 kDa NM OO6695 RaP2 interacting protein 8 NM OOO977 ribosomal protein L13 NM 002948 ribosomal protein L15 NM OOO994 ribosomal protein L32 NM OOO997 ribosomal protein L37 NM OOO998 ribosomal protein L37a NM 198486 ribosomal protein L7-like 1 NM OO2950 ribophorin I precursor P14 NM 007042 ribonuclease P 14 kDa subunit RM NM O19845 reprimo, TP53 dependant G2 arrest mediator PS23 NM 001025 ribosomal protein S23 PS6KA1 NMOO1OO6665 ribosomal protein S6 kinase, 90 kDa, polypeptide PS6KA2 NM OO1 OO6932 ribosomal protein S6 kinase, 90 kDa, polypeptide PS6KA3 NM OO4586 ribosomal protein S6 kinase, 90 kDa, polypeptide PS6KA4 NM 001006944 ribosomal protein S6 kinase, 90 kDa, polypeptide PS6KAS NM OO4755 ribosomal protein S6 kinase, 90 kDa, polypeptide RH NM OO6583 Peropsin RM2 NM 001034 ribonucleotide reductase M2 polypeptide RN3 NM 018427 RRN3 RNA polymerase I transcription factor RSAD2 NM 080657 radical S-adenosyl methionine domain containing RSBN1 NM 018364 round spermatid basic protein 1 RSL1D1 NM O15659 ribosomal L1 domain containing 1 RSNL2 NM O24692 restin-like 2 RSPO2 NM 178565 R-spondin family, member 2 RSPO4 NM 001029871 R-spondin family, member 4 isoform 1 precursor RSU1 NM 012425 ras Suppressor protein 1 isoform 1 RTF1 NM O15138 Paf1/RNA polymerase II complex component RTN2 NM 206902 reticulon 2 isoform D RTP1 NM 153708 receptor transporting protein 1 RTP4 NM 022147 28 kD interferon responsive protein RUNDC1 NM 173079 RUN domain containing 1 RUNDC2A NM 032167 RUN domain containing 2A RUNX1 NM 001001890 runt-related transcription factor 1 isoform b RUNX2 NM 001015051 runt-related transcription factor 2 isoform b RUNX3 NM OO1031680 runt-related transcription factor 3 isoform 1 RXRG NM OO6917 retinoid X receptor, gamma isoform a S100A16 NM 080388 S100 calcium binding protein A16 S100A4 NM OO2961 S100 calcium-binding protein A4 S1OOPBP NM 001017406 S100P binding protein Riken isoform b SACS NM O14363 Sacsin SAMD10 NM 080621 sterile alpha motif domain containing 10 SAMD12 NM 207506 sterile alpha motif domain containing 12 SAMD8 NM 144660 sterile alpha motif domain containing 8 SAMD9L. NM 152703 sterile alpha motif domain containing 9-like SAPS1 NM O14931 hypothetical protein LOC22870 SAPS2 NM 014678 hypothetical protein LOC9701 SAPS3 NM 018312 SAPS domain family, member 3 SAR1B NM OO 1033503 SAR1a gene homolog 2 SART1 NM OO5146 Squamous cell carcinoma antigen recognized by T SASH1 NM O15278 SAM and SH3 domain containing 1 SATB2 NM O15265 SATB family member 2 SATL1 NM 001012.980 spermidine?spermine N1-acetyltransferase-like SBK1 NM 001024401 SH3-binding domain kinase 1 SC4MOL NM 001017369 sterol-C4-methyl oxidase-like isoform 2 SCAMP1 NM 052822 Secretory carrier membrane protein 1 isoform 2 US 2009/0163434 A1 Jun. 25, 2009 48

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID S ymbol (Pruitt et al., 2005) Description S CAMP2 NM OO5697 Secretory carrier membrane protein 2 S CAMPS NM 138967 secretory carrier membrane protein 5 S CAND2 NM O22050 SCAN domain-containing protein 2 isoform 1 S CARAS NM 173833 hypothetical protein LOC286133 S CC-112 NM O15200 SCC-112 protein S CCPDH NM 016002 Saccharopine dehydrogenase (putative) S CIN NM 033128 Scinderin S CMH1 NM OO1031694 sex comb on midleg homolog 1 isoform 1 S CML2 NM OO6089 sex comb on midleg-like 2 S CN11A NM 014139 Sodium channel, voltage-gated, type XI, alpha S CN2B NM OO4588 Sodium channel, voltage-gated, type II, beta S CN3A NM OO6922 Sodium channel, voltage-gated, type III, alpha S CRN3 NM O24583 Secernin 3 S CRT2 NM 033129 Scratch 2 protein S DC2 NM OO2998 Syndecan 2 precursor DPR NM 004657 serum deprivation response protein EC14L2 NM 012429 SEC14-like 2 EC31L2 NM 1981.38 S. cerevisiae SEC31-like 2 isoform b EL1L. NM 005065 sel-1 Suppressor of lin-12-like ELE NM 000450 Selectin E precursor ELI NM 033505 Selenoprotein I ELPLG NM OO3OO6 Selectin P ligand EMA3E NM 012431 semaphorin 3E EMA4B NM 020210 Semaphorin 4B precursor EMA4G NM O17893 Semaphorin 4G EMASA NM OO3966 Semaphorin 5A ENP1 NMO14554 sentrin/SUMO-specific protease 1 ENP8 NM 145204 SUMO?sentrin specific protease family member 8 EPT11 NM 018243 Septin 11 EPT2 NM 001008491 Septin 2 EPT6 NM O15129 septin 6 isoform B ERF1A NM O21967 Small EDRK-rich factor 1A, telomeric ERF1B NM 022978 Small EDRK-rich factor 1B, centromeric ERINC1 NM 020755 tumor differentially expressed 2 ERP1 NM O14445 stress-associated endoplasmic reticulum protein ERPINB8 NM 002640 serine (or cysteine) proteinase inhibitor, clade ERPINE1 NM OOO602 plasminogen activator inhibitor-1 ERTAD2 NM 014755 SERTA domain containing 2 ESN1 NM O14454 sestrin 1 ESN2 NM 031459 sestrin 2 ET NM OO3011 SET translocation (myeloid leukemia-associated) ETD2 NM 014159 huntingtin interacting protein B ETD4 NM OO1007258 hypothetical protein LOC54093 isoform b EZ6 NM 178860 seizure related 6 homolog NM 182812 splicing factor 4 isoform c NM 001005158 Scm-like with four mbt domains 1 NM OO3012 secreted frizzled-related protein 1 NM OO3013 secreted frizzled-related protein 2 precursor NM 001017392 splicing factor, arginine?serine-rich 14 NM OO3O16 splicing factor, arginine?serine-rich 2 NM 199344 SFT2 domain containing 2 NM 178858 sideroflexin 2 NM 144579 sideroflexin 5 NM OOO337 delta-sarcoglycan isoform 1 NM OO1033578 serum glucocorticoid regulated kinase 3 isoform NM OO3901 sphingosine-1-phosphate lyase 1 NM OO5490 SH2 domain containing 3A NM 031469 SH3 domain binding glutamic acid-rich protein NM 001018009 SH3-domain binding protein 5 (BTK-associated) NM OO3O27 SH3-domain GRB2-like 3 NM 153271 SH3 and PX domain containing 3 NM 014631 SH3 multiple domains 1 NM 152550 SH3 domain containing ring finger 2 NM 012309 SH3 and multiple ankyrin repeat domains 2 NM O24745 SHC SH2-domain binding protein 1 NM 001010846 Src homology 2 domain containing E HF NM 138356 hypothetical protein LOC90525 HMT2 NM 005412 serine hydroxymethyltransferase 2 HOC2 NM OO7373 Soc-2 Suppressor of clear homolog AE NM 170601 cytosolic sialic acid 9-O-acetylesterase US 2009/0163434 A1 Jun. 25, 2009 49

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description NM O17699 SID1 transmembrane family, member 1 NM 03.3130 sialic acid binding Ig-like lectin 10 NM 052884 sialic acid binding Ig-like lectin 11 NM O25073 Suppressor of IKK epsilon NM OO9586 single-minded homolog 2 short isoform NM O15260 SIN3 homolog B, transcription regulator NM O15073 signal-induced proliferation-associated 1 like NM 001017524 sirtuin 3 isoform b NM O16538 sirtuin 7 NM OO3O36 v-ski sarcoma viral oncogene homolog NM 021181 SLAM family member 7 NM 000578 SO ute carrier fami 1 (proton-coupled NM OO6598 SO ute carrier fami 2 (potassium chloride NM 022444 SO ute carrier fami 3 (sodium/sulfate NM O15865 RACH1 NM 007163 SO ute carrier fami 4 (urea transporter), NM 213606 SO e carrier fami 6 (monocarboxylic acid NM 152527 SO e carrier fami 6 (monocarboxylic acid NM O06517 SO e carrier fami 6, member 2 NM OO4731 SO e carrier fami 6, member 7 NM 194298 SO e carrier fami 6 (monocarboxylic acid NM 012434 SO e carrier fami 7 (anion sugar NM 020309 SO e carrier fami 7, member 7 NM 139319 SO e carrier fami 7 (sodium-dependent NM O25243 SO e carrier fami 9, member 3 NM OO4171 SO e carrier fami , member 2 NMOO3038 SO e carrier fami , member 4 NM 018420 SO e carrier family 22 (organic cation NM OO3058 SO e carrier family 22 member 2 isoform a NM O21977 SO e carrier family 22 member 3 NM OO3060 SO e carrier family 22 member 5 NM 144712 SO e carrier family 23 (nucleobase NM 020689 SO e carrier family 24 NM 153646 SO e carrier family 24 member 4 isoform 1 NM 012140 SO e carrier family 25 (mitochondrial carrier; NM O14251 SO e carrier family 25, member 13 (citrin) NM 013386 SO e carrier family 25 member 24 isoform 1 NM OO4277 SO e carrier family 25, member 27 NM 207348 SO e carrier family 25, member 34 NM 000441 Pe NM 052832 SO e carrier family 26, member 7 isoform a NM OO1532 SO e carrier family 29 (nucleoside NM O3O807 glucose transporter protein 10 isoform a NM OOO340 SO ute carrier family 2 (facilitated glucose NM OO6931 SO ute carrier family 2 (facilitated glucose NM 001042 glucose transporter 4 NM 020062 S L 2A4 regulator NM OO3O39 e carrier family 2 (facilitated NM 017585 e carrier family 2 (facilitated glucose NM 001004433 e carrier family 30 (zinc transporter), NM OO3459 e carrier family 30 (zinc transporter), NM 133496 transporter like 2 NM OO1859 e carrier family 31 (copper transporters), NM O17945 e carrier family 35, member A5 NM 032826 e carrier family 35, member B4 NM O24881 e carrier family 35, member E1 NM 018656 e carrier family 35, member E2 NM 173508 e carrier family 35, member F3 NM O25181 e carrier family 35, member F5 NM O78483 e carrier family 36 member 1 NM 181776 e carrier family 36 (protonamino acid NM OO1467 e carrier family 37 (glycerol-6-phosphate NM 014585 SO e carrier family 40 (iron-regulated NM 173854 SO e carrier family 41 member 1 NM O25257 NG22 protein isoform 1 NM 001012.509 membrane-associated transporter protein isoform NM OO3615 SO ute carrier family 4, Sodium bicarbonate NM 178498 SO ute carrier family 5 (sodium glucose NM 021815 SO ute carrier family 5 (choline transporter), NM OO5629 SO ute carrier family 6 (neurotransmitter US 2009/0163434 A1 Jun. 25, 2009 50

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description NM 001024845 solute carrier family 6 member 9 isoform 3 NM O14331 Solute carrier family 7, (cationic amino acid NM OO1008539 solute carrier family 7, member 2 isoform 1 NM OO3983 Solute carrier family 7 (cationic amino acid NM OO3O48 Solute carrier family 9 (sodium hydrogen NM OO4252 Solute carrier family 9 (sodium hydrogen NM OO6359 Solute carrier family 9 (sodium hydrogen NM O15266 Na+ H+ exchanger isoform 8 NM 173653 Solute carrier family 9 (sodium hydrogen NM O17435 Solute carrier organic anion transporter family, NM 180991 Solute carrier organic anion transporter family, NM 032336 SLDS NM 032539 SLIT and NTRK-like family, member 2 NM O14926 slit and trk like 3 protein NM 014720 serine threonine kinase 2 S LTM NM 00101.3843 modulator of estrogen induced transcription SMA4 NM 021652 SMA4 SMAS NM 021036 SMAS SMAD1 NM 001003688 Sma- and Mad-related protein 1 SMAD2 NM 001003652 Sma- and Mad-related protein 2 SMADS NM 001001419 SMAD, mothers against DPP homolog 5 SMAD6 NM OO5585 MAD, mothers against decapentaplegic homolog 6 SMAD7 NM OO5904 MAD, mothers against decapentaplegic homolog 7 SMC1L1 NM OO6306 SMC1 structural maintenance of SMC1L2 NM 148674 SMC1 structural maintenance of chromosomes SMC4L1 NM OO1002799 SMC4 structural maintenance of chromosomes SMEK2 NM020463 hypothetical protein LOC57223 SMG NM O15092 PI-3-kinase-related kinase SMG-1 SMOC1 NM 0221.37 Secreted modular calcium-binding protein 1 SMOC2 NM 022138 Secreted modular calcium-binding protein 2 SMYD1 NM 1982.74 SET and MYND domain containing 1 SMYD4 NM 052928 SET and MYND domain containing 4 SNAP23 NM 003825 synaptosomal-associated protein 23 isoform SNAPC4 NM OO3O86 Small nuclear RNA activating complex, SNF1LK NM 173354 SNF1-like kinase SNPH NM 014723 Syntaphilin SNRK NM O17719 SNF related kinase SNRPD3 NM OO4175 Small nuclear ribonucleoprotein polypeptide D3 SNX11 NM 013323 Sorting nexin 11 SNX16 NM 0221.33 Sorting nexin 16 isoform a SNX19 NM 014758 Sorting nexin 19 SNX22 NM O24798 Sorting nexin 22 SNX27 NM 030918 Sorting nexin family member 27 SNX9 NM O16224 Sorting nexin 9 SOCS6 NM OO4232 Suppressor of cytokine signaling 6 SOD2 NM 000636 manganese Superoxide dismutase isoform A SOLH NM OO5632 Small optic lobes SORBS2 NM OO3603 Sorbin and SH3 domain containing 2 isoform 1 SORL1 NM OO3105 Sortilin-related receptor containing LDLR class SORT1 NM OO2959 Sortilin 1 preproprotein SOX1 NM OO5986 SRY (sex determining region Y)-box 1 SOX12 NM OO6943 SRY (sex determining region Y)-box 12 SOX4 NM OO3107 SRY (sex determining region Y)-box 4 SOX7 NM 031439 SRY-box 7 P140 NM OO7237 SP140 nuclear body protein isoform 1 P4 NM 003112 Sp4 transcription factor NM 199262 Sp6 transcription factor P8 NM 1827OO Sp8 transcription factor isoform 1 PACA1 NM 030960 sperm acrosome associated 1 PACA4 NM 133498 sperm acrosomal membrane protein 14 PARC NM OO3118 Secreted protein, acidic, cysteine-rich PATA13 NM 153023 spermatogenesis associated 13 PATA3 NM 139073 testis and spermatogenesis cell apoptosis PATS2 NM 023071 spermatogenesis associated, serine-rich 2 PBC24 NM 182513 spindle pole body component 24 homolog PECC1 NM OO1033553 spectrin domain with coiled-coils 1 NSP5b3b PFH1 NM OO6459 SPFH domain family, member 1 PFH2 NM 007175 SPFH domain family, member 2 isoform 1 PG20 NM O15087 Spartin PIRE2 NM 032451 spire homolog 2 US 2009/0163434 A1 Jun. 25, 2009 51

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description PN NM OO1030288 Sialophorin POCK1 NM 004598 sparcosteonectin, cwcv and kazal-like domains POCK2 NM 014767 sparcosteonectin, cwcv and kazal-like domains PRN NM 001012508 shadow of prion protein PRR2B NM 001017418 Small proline-rich protein 2B PRR2E NM 001024209 Small proline-rich protein 2E PRR2F NM 001014450 Small proline-rich protein 2F PRY4. NM 030964 sprouty homolog 4 PSB4 NM 08O862 SPRY domain-containing SOCS box protein SSB-4 PTBN4 NM 020971 spectrin, beta, non-erythrocytic 4 isoform 1 PTLC1 NM 178324 serine palmitoyltransferase subunit 1 isoform b PTLC2 NM 004.863 serine palmitoyltransferase, long chain base PTY2D1 NM 194285 hypothetical protein LOC144108 STM1 NM OO3900 sequestosome 1 D5A2L2 NM 001010874 5 alpha-reductase 2-like 2 GAP3 NM OO1033116 SLIT-ROBO Rho GTPase activating protein 3 NM OO3130 Sorcin isoform a P72 NM OO6947 signal recognition particle 72 kDa PK1 NM OO3137 SFRS protein kinase PK2 NM 182691 SFRS protein kinase 2 isoform b S RXN1 NM 080725 Sulfiredoxin 1 homolog SS18L1 NM O15558 SS18-like protein 1 SSBP3 NM 001009955 single stranded DNA binding protein 3 isoform c SSFA2 NM OO6751 sperm specific antigen 2 SSH2 NM 03.3389 slingshot 2 SSPN NM 005086 Sarcospan SSR3 NM 007107 signal sequence receptor gamma Subunit SSTR2 NM 001050 Somatostatin receptor 2 SSX2IP NM 014021 synovial sarcoma, X breakpoint 2 interacting ST3GAL1 NM OO3O33 sialyltransferase 4A ST6GAL1 NM OO3O32 sialyltransferase 1 isoform a ST6GALNAC3 NM 152996 ST6 ST6GALNAC6 NM 013443 ST6 ST8SLA2 NM OO6011 ST8 alpha-N-acetyl-neuraminide ST8SIA4 NM OO5668 ST8 alpha-N-acetyl-neuraminide STAC NM OO3149 SH3 and cysteine rich domain STAC2 NM 1989.93 SH3 and cysteine rich domain 2 STAM2 NM 005843 signal transducing adaptor molecule 2 STAR NM OOO349 steroidogenic acute regulator isoform 1 STARD8 NM 014725 START domain containing 8 STAT1 NM OO7315 signal transducer and activator of transcription STAT3 NM OO3150 signal transducer and activator of transcription STATSB NM 012448 signal transducer and activator of transcription STC1 NM OO3155 Stanniocalcin 1 precursor STIM1 NM OO3156 stromal interaction molecule 1 precursor STIM2 NM 020860 stromal interaction molecule 2 STK11 NM 000455 serine threonine protein kinase 11 STK11P NM 052902 LKB1 interacting protein STK17B NM 004226 serine threonine kinase 17b STK33 NM 030906 serine threonine kinase 33 STK38 NM OO7271 serine threonine kinase 38 STK4 NM OO6282 serine threonine kinase 4 STOM NM 004.099 stomatin isoform a STRBP NM 018387 spermatid perinuclear RNA-binding protein STRN3 NM 014574 nuclear autoantigen STS-1 NM 032873 Cbl-interacting protein Sts-1 STX6 NM OO5819 Syntaxin 6 STYX NM 145251 serine threonine tyrosine interacting protein SUDS3 NM 022491 Suppressor of defective silencing 3 SUHW2 NM 080764 Suppressor of hairy wing homolog 2 SUHW3 NM O17666 Suppressor of hairy wing homolog 3 SUHW4 NM OO1002843 Suppressor of hairy wing homolog4 isoform 2 SULF1 NM O15170 Sulfatase 1 SULT2A1 NM OO3167 Sulfotransferase family, cytosolic, 2A, SUMF1 NM 182760 Sulfatase modifying factor 1 SUPTTL NM 014.860 SPTF-associated factor 65 gamma SUSD1 NM 022486 Sushi domain containing 1 SUV39H2 NM O24670 Suppressor of variegation 3-9 homolog 2 SUV42OH1 NM O17635 Suppressor of variegation 4-20 homolog 1 isoform SVEH NM 03.1905 SVH protein US 2009/0163434 A1 Jun. 25, 2009 52

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description SWAP70 NM O15055 SWAP-70 protein SYAP1 NM 032796 SYAP1 protein SYNE1 NM O15293 nesprin 1 isoform beta SYNE2 NM O1518O spectrin repeat containing, 2 SYNGR2 NM 004710 synaptogyrin 2 SYN2BP NM 018373 synaptoanin 2 binding protein SYNPO2 NM 133477 synaptopodin 2 SYNPO2L NM O24875 synaptopodin 2-like SYT10 NM 1989.92 synaptotagmin 10 SYT13 NM 020826 synaptotagmin XIII SYT15 NM 03.1912 synaptotagmin XV isoform a SYT7 NM 004200 synaptotagmin VII SYTL4 NM 080737 synaptotagmin-like 4 (granuphilin-a) TACC1 NM OO6283 transforming, acidic coiled-coil containing TAF7 NM 005642 TATA box-binding protein-associated factor 2F TAF9B NM O15975 transcription associated factor 9B TAGAP NM 054114 T-cell activation Rho GTPase-activating protein TAIP-2 NM 024969 TGF-beta induced apoptosis protein 2 TAL NM OO3189 T-cell acute lymphocytic leukemia 1 TANC1 NM 03.3394 TPR domain, ankyrin-repeat and TAOK2 NM O16151 TAOkinase 2 isoform 2 TAOK3 NM 016281 TAOkinase 3 TAPBP NM 172208 tapasin isoform 2 precursor TAT NM 000353 tyrosine aminotransferase TAX1BP1 NM OO6024 ax1 (human T-cell leukemia virus type I) TBC1D1 NM O15173 TBC1 (tre-2/USP6, BUB2, ccdc16) domain family, TBC1D1 OC NM 198517 TBC1 domain family, member 10C TBC1D15 NM O22771 TBC1 domain family, member 15 TBC1D17 NM O24682 TBC1 domain family, member 17 TBC1D2 NM 018421 TBC1 domain family, member 2 TBC1D4 NM O14832 TBC1 domain family, member 4 TBC1D8 NM 007063 TBC1 domain family, member 8 TBC1D9 NM O15130 hypothetical protein LOC23158 TBL1X NM 005647 transducin beta-like 1X TBRG1 NM 032811 transforming growth factor beta regulator 1 TBX19 NM OO5149 T-box 19 TBX3 NM OO5996 T-box 3 protein isoform 1 TCEAL7 NM 152278 hypothetical protein LOC56849 TCEB3 NM OO31.98 elongin A TCF21 NM OO32O6 transcription factor 21 TCF7 NM OO32O2 transcription factor 7 (T-cell specific, TCF7L1 NM 031283 HMG-box transcription factor TCF-3 TCL6 NM O14418 T-cell leukemia/lymphoma 6 isoform TCL6a2 TCN2 NM OOO355 transcobalamin II precursor TCOF1 NM OO10O8657 Treacher Collins-Franceschetti syndrome 1 TCTA NM 022171 T-cell leukemia translocation altered gene TCTEX1D1 NM 152665 hypothetical protein LOC200132 TDRD1 NM 198795 tudor domain containing 1 TEGT NM O03217 estis enhanced gene transcript (BAX inhibitor TEK NM 000459 TEK tyrosine kinase, endothelial precursor TEP1 NM 007110 elomerase-associated protein 1 TESC NM O17899 Tescalcin TEX14 NM 031272 estis expressed sequence 14 isoform b TEX15 NM 031271 estis expressed sequence 15 TEX261 NM 144582 estis expressed sequence 261 TFEC NM 001018058 transcription factor EC isoform b TGFB11 NM O15927 androgen receptor coactivator ARA55 TGFB2 NM O03238 transforming growth factor, beta 2 TGFBI NM OOO358 transforming growth factor, beta-induced, 68 kDa TGFBR2 NM 001024847 TGF-beta type II receptor isoform A precursor TGM2 NM 198951 2 isoform b TGM3 NM OO3245 transglutaminase 3 precursor TGOLN2 NM OO6464 trans-golgi network protein 2 THADA NM 198554 hyroid adenoma associated isoform 2 THAP2 NM 031435 THAP domain containing, apoptosis associated THAP6 NM 144721 THAP domain containing 6 THEBD NM OOO361 hrombomodulin precursor THEBS2 NM OO3247 hrombospondin 2 precursor THEDC1 NM 018324 hioesterase domain containing 1 isoform 1 THEM4 NM 053055 hioesterase Superfamily member 4 isoform a US 2009/0163434 A1 Jun. 25, 2009 53

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description THEMS NM 182578 hioesterase Superfamily member 5 THEX1 NM 153332 histone mRNA 3’ end-specific exonuclease THRA NM 1993.34 hyroid hormone receptor, alpha isoform 1 THSD3 NM 182SO9 hrombospondin, type I domain containing 3 THUMPD1 NM O17736 THUMP domain containing 1 TIFA NM OS2864 TRAF-interacting protein with a TIMM10 NM O12456 translocase of inner mitochondrial membrane 10 TIMM17A NM OO633S translocase of inner mitochondrial membrane 17 TIMM44 NM OO6351 translocase of inner mitochondrial membrane 44 TIMMSO NM OO1 OO1563 translocase of inner mitochondrial membrane 50 TIMP2 NM OO3255 issue inhibitor of metalloproteinase 2 TIMP3 NM OOO362 issue inhibitor of metalloproteinase 3 TIPARP NM O15508 TCDD-inducible poly(ADP-ribose) polymerase TJP1 NM OO3257 ight junction protein 1 isoform a TLE4 NM OO7005 transducin-like enhancer protein 4 NM O12464 olloid-like 1 NM OO3262 translocation protein 1 NM O16S 62 ol-like receptor 7 NM OO 102438O TM2 domain containing 2 isoform b NM O16056 transmembrane BAX inhibitor motif containing 4 NM O24847 transmembrane channel-like 7 NM OO 101739S transmembrane and coiled-coil domains 1 isoform NM O2O698 transmembrane and coiled-coil domains 3 NM O16040 transmembrane emp24 protein transport domain NM OO1 OO1723 transmembrane protein 1 isoform b NM 178520 hypothetical protein LOC284186 NM O2S222 hypothetical protein PRO2730 NM OS2932 pro-oncosis receptor inducing membrane injury NM O17849 hypothetical protein LOC55654 NM 152913 hypothetical protein LOC222865 NM 032021 hypothetical protein LOC83935 NM 022918 hypothetical protein LOC65084 NM 016464 hypothetical protein LOC51524 NM 213599 transmembrane protein 16E NM 001025356 transmembrane protein 16F NM 198276 transmembrane protein 17 NM 152834 transmembrane protein 18 NM 147156 phosphatidylcholine:ceramide NM 032780 transmembrane protein 25 NM 178505 transmembrane protein 26 NM O15686 transmembrane protein 28 NM 018247 transmembrane protein 30A NM 024074 transmembrane protein 38A NM 018306 transmembrane protein 40 NM 080652 transmembrane protein 41A transmembrane protein 45B transmembrane protein 5 OB transmembrane protein 56 transmembrane protein 64 hypothetical protein LOC137835 hypothetical protein LOC128338 hypothetical protein LOC283232 hypothetical protein LOC145978 tropomodulin 1 tropomodulin 2 (neuronal) hymopoietin isoform beta TMPRSS11B transmembrane protease, serine 11B TMTC2 hypothetical protein LOC160335 TNFAIP1 tumor necrosis factor, alpha-induced protein 1 TNFAIP3 tumor necrosis factor, alpha-induced protein 3 TNFAIP8L1 NM S2362 tumor necrosis factor, alpha-induced protein TNFAIP8L2 NM O24575 tumor necrosis factor, alpha-induced protein TNFAIP8L3 NM 207381 tumor necrosis factor, alpha-induced protein TNFRSF1OA NM 003844 tumor necrosis factor receptor Superfamily, TNFRSF10B NM OO3842 tumor necrosis factor receptor Superfamily, TNFRSF1OD NM OO3840 tumor necrosis factor receptor Superfamily, TNFRSF17 NM OO1192 tumor necrosis factor receptor Superfamily, TNFRSF19 NM 148957 tumor necrosis factor receptor Superfamily, TNFRSF1B NM 001066 tumor necrosis factor receptor 2 precursor TNFRSF21 NM O14452 tumor necrosis factor receptor Superfamily, US 2009/0163434 A1 Jun. 25, 2009 54

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description TNFSF11 NM 003701 tumor necrosis factor ligand Superfamily, member TNFSF14 NM OO3807 tumor necrosis factor ligand Superfamily, member TNFSF15 NM OO5118 tumor necrosis factor (ligand) Superfamily, TNIP3 NM O24873 hypothetical protein LOC79931 TNK2 NM 001010938 tyrosine kinase, non-receptor, 2 isoform 2 TNKS1BP1 NM 03.3396 ankyrase 1-binding protein of 182 kDa TNKS2 NM O25235 ankyrase, TRF1-interacting ankyrin-related TNNI1 NM OO3281 troponin I, skeletal, slow TNRC6A NM O14494 Erinucleotide repeat containing 6A isoform 1 TNRC6B NM 001024843 Erinucleotide repeat containing 6B isoform 2 TOLLIP NM O19009 oll interacting protein TOMM4OL NM 032174 translocase of outer mitochondrial membrane 40 TOMMT NM O19059 6.2 kd protein TOMM7OA NM O14820 translocase of outer mitochondrial membrane 70 TOP3A NM 004618 opoisomerase (DNA) III alpha TOPORS NM OO58O2 opoisomerase I binding, arginine?serine-rich TOR1B NM 014506 orsin family 1, member B (torsin B) TP53INP1 NM 033285 tumor protein p53 inducible nuclear protein 1 TP53INP2 NM 021202 tumor protein p53 inducible nuclear protein 2 TPD52 NM 001025252 tumor protein D52 isoform 1 TPK1 NM 022445 hiamin pyrophosphokinase 1 TPM4 NM OO3290 tropomyosin 4 TRA16 NM 176880 TR4 orphan receptor associated protein TRA16 TRAK1 NM O14965 OGT(O-Glc-NAc transferase)-interacting protein TRAM1 NM O14294 translocating chain-associating membrane TRAM2 NM 012288 translocation-associated membrane protein 2 TRAPPC2 NMOO1011658 trafficking protein particle complex 2 AD3 NM O19011 TRIAD3 protein isoform c AP1 NM 016399 p53-inducible cell-survival factor B3 NM 021158 ribbles 3 M10 NM 052828 tripartite motif-containing 10 isoform 2 M2 NM O15271 tripartite motif-containing 2 M22 NM OO6074 tripartite motif-containing 22 M26 NM 003449 tripartite motif-containing 26 M3 NM OO6458 tripartite motif-containing 3 M31 NM 052816 tripartite motif protein 31 isoform beta M32 NM 012210 TAT-interactive protein, 72-KD M33 NM O15906 tripartite motif-containing 33 protein isoform M36 NM 018700 tripartite motif-containing 36 isoform 1 M37 NM O15294 tripartite motif-containing 37 protein M4 NM 033017 tripartite motif protein TRIM4 isoform alpha M55 NM 033058 ring finger protein 29 isoform 2 MS6 NM 030961 tripartite motif-containing 56 MS8 NM O15431 tripartite motif-containing 58 M6S NM 173547 tripartite motif-containing 65 M68 NM 018073 ring finger protein 137 M7 NM 203293 tripartite motif-containing 7 isoform 1 M8 NM 030912 tripartite motif-containing 8 M9 NM 052978 tripartite motif protein 9 isoform 2 P10 NM 004240 hyroid hormone receptor interactor 10 P11 NM OO4239 hyroid hormone receptor interactor 11 TRMU NM OO10O8568 RNA 5-methylaminomethyl-2-thiouridylate PA1 NM O07332 ankyrin-like protein 1 PC1 NM OO3304 transient receptor potential cation channel, PC4 NM O16179 transient receptor potential 4 PM1 NM OO2420 transient receptor potential cation channel, PM6 NM O17662 transient receptor potential cation channel, PS1 NM 014112 Zinc finger transcription factor TRPS1 PV6 NM 018646 transient receptor potential cation channel, TRSPAP1 NM O17846 RNA selenocysteine associated protein TRUB1 NM 1391.69 TruB pseudouridine (psi) synthase homolog 1 TSC22D2 NM 014779 TSC22 domain family 2 TSCOT NM 033051 hymic stromal co-transporter TSEN2 NM O25265 RNA splicing endonuclease 2 homolog TSG101 NM OO6292 tumor Susceptibility gene 101 TSHZ3 NM 020856 Zinc finger protein 537 TSNAX NM OO5999 translin-associated factor X TSPAN14 NM 030927 etraspanin 14 TSPAN17 NM 001006616 transmembrane 4 superfamily member 17 isoform c TSPAN4 NM 001025234 etraspanin 4 isoform a US 2009/0163434 A1 Jun. 25, 2009 55

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description TSPAN9 NM OO6675 etraspanin 9 TSPYL1 NM OO3309 TSPY-like 1 TSR1 NM 018128 hypothetical protein LOC55720 TTF2 NM 003594 transcription termination factor, RNA polymerase TTL NM 153712 tubulin tyrosine ligase TTLL6 NM 173623 hypothetical protein LOC284076 TTN NM 003319 itin isoform N2-B TULP3 NM OO3324 tubby like protein 3 TUSC2 NM 007275 tumor Suppressor candidate 2 TWIST1 NM 000474 Twist TXLNA NM 175852 Taxillin TXNDC10 NM O19022 hioredoxin domain containing 10 TXNDC4 NM O15051 hioredoxin domain containing 4 (endoplasmic TXNIP NM OO6472 hioredoxin interacting protein TXNL2 NM OO6541 hioredoxin-like TYRP1 NM 000550 tyrosinase-related protein 1 UACA NM 0010O8224 uveal autoantigen with coiled-coil domains and BAP1 NM O16525 biquitin associated protein 1 BASH3A NM 001001895 biquitin associated and SH3 domain containing, BC NM 021009 biquitin C BE2B NM OO3337 biquitin-conjugating enzyme E2B BE2G1 NM OO3342 biquitin-conjugating enzyme E2G 1 isoform 1 BE2G2 NM OO3343 biquitin-conjugating enzyme E2G 2 isoform 1 BE21 NM 016021 biquitin-conjugating enzyme E2, J1 BE2O2 NM 173469 biquitin-conjugating enzyme E2O (putative) 2 BE2W NM 001001481 ypothetical protein LOC55284 isoform 1 BE3A NMOOO462 biquitin protein ligase E3A isoform 2 BE3C NM 014671 biquitin protein ligase E3C BE4A NM OO4788 biquitination factor E4A BL4A NM O14235 ubiquitin-like 4 BOXS NM O14948 U-box domain containing 5 isoform a BQLN1 NM 013438 ubiquilin 1 isoform 1 BQLN4 NM O2O131 ataxin-1 ubiquitin-like interacting protein BXD4 NM 181713 UBX domain containing 4 BXD8 NM 014613 UBX domain containing 8 CP3 NM OO3356 uncoupling protein 3 isoform UCP3L EV3 NM 018314 ubiquitin-conjugating enzyme E2-like GDH NM OO3359 UDP-glucose dehydrogenase GP2 NM 001001521 UDP-glucose pyrophosphorylase 2 isoform b LK1 NM 003565 unc-51-like kinase 1 UNC5C NM 003728 UNCSCL NM 173561 unc-5 homolog C-like UNC93B1 NM 030930 unc-93 homolog B1 UNQ9370 NM 207447 hypothetical protein LOC400454 UPF3A NM 023011 UPF3 regulator of nonsense transcripts homolog A UPK1A NM 007000 uroplakin 1A UPK1B NM OO6952 uroplakin 1B UPP1 NM OO3364 uridine phosphorylase 1 UQCRB NM OO6294 ubiquinol-cytochrome c reductase binding URB NM 199511 steroid-sensitive protein 1 URG4 NM O17920 hypothetical protein LOC55665 USP14 NM O05151 ubiquitin specific protease 14 isoform a USP25 NM 013396 ubiquitin specific protease 25 USP28 NM 020886 ubiquitin specific protease 28 USP3 NM OO6537 ubiquitin specific protease 3 USP32 NM 032582 ubiquitin specific protease 32 USP33 NM O15017 ubiquitin specific protease 33 isoform 1 USP37 NM 020935 ubiquitin specific protease 37 USP46 NM O22832 ubiquitin specific protease 46 USP47 NM O17944 ubiquitin specific protease 47 USP49 NM 018561 ubiquitin specific protease 49 USP9Y NM 004654 ubiquitin specific protease 9, Y-linked UST NM OO5715 uronyl-2-sulfotransferase UTP14C NM 021645 UTP14, U3 small nucleolar ribonucleoprotein, UXS1 NM O25076 UDP-glucuronate decarboxylase 1 WANGL1 NM 138959 vang-like 1 WAPA NM 003574 vesicle-associated membrane protein-associated WASP NM OO10O8736 vasodilator-stimulated phosphoprotein isoform 2 WAV2 NM OO3371 vaV 2 oncogene WAX1 NM 1991.31 ventral anterior homeobox 1 US 2009/0163434 A1 Jun. 25, 2009 56

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Transcript ID (Pruitt et al., 2005) Description NM OO3373 winculin isoform VCL NM OO3374 voltage-dependent anion channel 1 NM 001025366 vascular endothelial growth factor isoform a NM 017599 transmembrane protein vezatin NM 153453 vestigial-like 2 isoform 2 NM 0162O6 colon carcinoma related protein NM 014667 vestigial like 4 NM 000551 von Hippel-Lindau tumor suppressor isoform 1 NM 001018056 very low density lipoprotein receptor isoform b MP NM 080723 vesicular membrane protein p24 NM O17684 vacuolar protein sorting 13C protein isoform 1A NM O15378 vacuolar protein sorting 13D isoform 1 NM 001005753 vacuolar protein sorting 24 isoform 2 NM 032353 vacuolar protein sorting 25 NM 004.896 vacuolar protein sorting 26 homolog A isoform 1 NM 016075 vacuolar protein sorting 36 NM O17966 vacuolar protein sorting 37C NM 004.869 vacuolar protein sorting factor 4B NM OO3384 vaccinia related kinase 1 NM 1826O7 V-set and immunoglobulin domain containing 1 NM 014588 visual system homeobox 1 protein isoform a NM 016628 WW domain-containing adapter with a coiled-coil NM OO6990 WAS protein family, member 2 NM OO6646 WAS protein family, member 3 NM OO3941 Wiskott-Aldrich syndrome gene-like protein NM 022170 eukaryotic translation initiation factor 4H NM 017528 Williams Beuren syndrome chromosome region 22 NM 014991 D repeat and FYVE domain containing 3 isoform NM OO5112 D repeat-containing protein 1 isoform 2 NM 170710 D repeat domain 17 isoform 1 NM O25132 D repeat domain 19 NM 152418 pothetical protein LOC138009 NM O25230 D repeat domain 23 isoform 1 NM O25160 D repeat domain 26 NM 024345 D repeat domain 32 NM OO1006623 D repeat domain 33 isoform 3 NM OO1 OO6657 D repeat domain 35 isoform 1 NM 139281 D repeat domain 36 NM 014023 D repeat domain 37 NM 004804 D repeat domain 39 NM 018669 D repeat domain 4 protein NM 178470 D repeat domain 40B NM O15726 326 NM 020839 D repeat domain 48 NM O19069 D repeat domain 5B NM 032856 D repeat domain 73 NM OO3390 wee1 tyrosine kinase NM OO6005 Wolframin NM OO7331 Wolf-Hirschhorn syndrome candidate 1 protein NM O22470 p53 target Zinc finger protein isoform 1 NM 133264 WIRE protein NM 003881 WNT1 inducible signaling pathway protein 2 NM OO6648 WNK lysine deficient protein kinase 2 NM OO10O2838 WNK lysine deficient protein kinase 3 isoform 2 NM OO3392 wingless-type MMTV integration site family, NM 058238 wingless-type MMTV integration site family, NM 018639 WD SOCS-box protein 2 NM OOO378 Wilms tumor 1 isoform A NM 199423 WW domain containing E3 ubiquitin protein ligase NM OO2995 chemokine (C motif) ligand 1 NM 003175 chemokine (C motif) ligand 2 NM 207411 XK-related protein 5a NM 212559 X Kell blood group precursor-related, X-linked NM 020750 exportin 5 NM 005431 X-ray repair cross complementing protein 2 NM O19001 5'-3' exoribonuclease 1 NM 138568 protein 7 transactivated by hepatitis B virus X NM 001012424 YY1-associated factor 2 isoform b NM O15936 tyrosyl-tRNA synthetase 2 (mitochondrial) NM 005433 viral oncogene yes-1 homolog 1 US 2009/0163434 A1 Jun. 25, 2009 57

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description YIPF5 NM 001024947 Smooth muscle cell associated protein 5 YME1L1 NM O14263 YME1-like 1 isoform 3 YOD1 NM 018566 hypothetical protein LOC55432 YPEL1 NM 013313 yippee-like 1 YPEL2 NM 0010054.04 yippee-like 2 YPEL4 NM 145008 yippee-like 4 YTHDF3 NM 152758 YTH domain family, member 3 YWHAQ NM OO6826 tyrosine 3 tryptophan 5-monooxygenase YWHAZ NM OO3406 tyrosine 3 tryptophan 5-monooxygenase ZADH1 NM 152444 Zinc binding alcohol dehydrogenase, domain ZADH2 NM 175907 Zinc binding alcohol dehydrogenase, domain A. K NM 133646 MLK-related kinase isoform 2 BED1 NM OO4729 Ac-like transposable element NM 030776 tumor stroma and activated macrophage protein BTB24 NM 014797 Zinc finger and BTB domain containing 24 BTB33 NM OO6777 BTB39 NM O14830 Zinc finger and BTB domain containing 39 BTB4 NM 020899 Zinc finger and BTB domain containing 4 BTB41 NM 194314 Zinc finger and BTB domain containing 41 BTB5 NM O14872 Zinc finger and BTB domain containing 5 BTB7A NM O15898 Zinc finger and BTB domain containing 7A BTB9 NM 152735 Zinc finger and BTB domain containing 9 C3H12B NM 001010888 hypothetical protein LOC340554 CCHC16 NM OO1004308 hypothetical protein LOC340595 DHHC1 NM 013304 Zinc finger, DHHC domain containing 1 HHC23 NM 173570 Zinc finger, DHHC domain containing 23 HHC9 NMOO10O8222 Zinc finger, DHHC domain containing 9 FP106 NM O22473 Zinc finger protein 106 homolog FP161 NM OO3409 Zinc finger protein 161 homolog FP30 NM O14898 Zinc finger protein 30 homolog FP42 NM 174900 Zinc finger protein 42 FP90 NM 133458 Zinc finger protein 90 homolog FP91 NM 053023 Zinc finger protein 91 isoform 1 FP95 NM 014569 Zinc finger protein 95 homolog FPM2 NM 012082 Zinc finger protein, multitype 2 FYVE20 NM O22340 FYVE-finger-containing Rab5 effector protein FYVE21 NM 024071 Zinc finger, FYVE domain containing 21 FYVE26 NM O15346 Zinc finger, FYVE domain containing 26 FYVE9 NM OO4799 Zinc finger, FYVE domain containing 9 isoform 3 HX2 NM O14943 Zinc fingers and homeoboxes 2 HX3 NM O15035 Zinc fingers and homeoboxes 3 NM 003412 Zinc finger protein of the cerebellum 1 NM 032153 Zinc finger protein of the cerebellum 4 M3 NM 052882 Zinc finger, imprinted 3 KSCAN 1 NM OO3439 Zinc finger protein 36 ZMYM6 NM 007167 Zinc finger protein 258 ZNF114 NM 153608 Zinc finger protein 114 ZNF134 NM OO3435 Zinc finger protein 134 ZNF136 NM OO3437 Zinc finger protein 136 (clone pHZ-20) ZNF137 NM OO3438 Zinc finger protein 137 (clone pHZ-30) ZNF14 NM 021030 Zinc finger protein 14 ZNF140 NM OO3440 Zinc finger protein 140 (clone pHZ-39) ZNF148 NM 021964 Zinc finger protein 148 (pHZ-52) ZNF155 NM OO3445 Zinc finger protein 155 ZNF160 NM 033288 Zinc finger protein 160 ZNF161 NM 0071.46 Zinc finger protein 161 ZNF177 NM OO3451 Zinc finger protein 177 ZNF18O NM 013256 Zinc finger protein 180 (HHZ168) ZNF187 NM 001023560 Zinc finger protein 187 ZNF192 NM OO6298 Zinc finger protein 192 ZNF195 NM 007152 Zinc finger protein 195 ZNF197 NM OO6991 Zinc finger protein 197 isoform 1 ZN NM 001017396 Zinc finger protein 2 isoform b ZNF2O2 NM OO3455 Zinc finger protein 202 ZNF213 NM 004220 Zinc finger protein 213 ZNF217 NM OO6526 Zinc finger protein 217 ZNF23 NM 145911 Zinc finger protein 23 ZNF236 NM OO7345 Zinc finger protein 236 ZNF238 NM OO6352 Zinc finger protein 238 isoform 2 ZNF239 NM OO5674 Zinc finger protein 239 US 2009/0163434 A1 Jun. 25, 2009 58

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description

F25 NM 145011 Zinc finger O ein 25 F264 NM OO3417 Zinc finger O ein 264 F271 NM OO6629 Zinc finger O ein 271 F28 NM OO6969 Zinc finger O ein 28 (KOX 24) F282 NM 003575 Zinc finger O ein 282 F295 NM 020727 Zinc finger O ein 295 F304 NM O2O657 Zinc finger O ein 304 F307 NM 019110 Zinc finger O ein 307 F31 NM 145238 Zinc finger O ein 31 F32O NM 207333 Zinc finger O ein 320 F329 NM O24620 Zinc finger O ein 329 F331 NM 018555 Zinc finger O ein 331 F333 NM 032433 Zinc finger O ein 333 F336 NM 022482 Zinc finger O ein 336 F337 NM O15655 Zinc finger O ein 337 F33A NM OO6974 Zinc finger O ein 33a F346 NM 012279 Zinc finger O ein 346 F347 NM 032584 Zinc finger O ein 347 F367 NM 153695 Zinc finger O ein 367 F385 NM O15481 Zinc finger O ein 385 F394 NM 032164 Zinc finger O ein 99 F398 NM 020781 Zinc finger 398 isoform b F417 NM 152475 Zinc finger O ein 417 F43 NM OO3423 Zinc finger O ein 43 (HTF6) F430 NM O251.89 Zinc finger O ein 430 F431 NM 133473 Zinc finger O ein 431 F440 NM 152357 Zinc finger O ein 440 F445 NM 181489 Zinc finger O ein 445 F452 NM 052923 Zinc finger O ein 452 F454 NM 182594 Zinc finger O ein 454 F468 NM OO10088O1 Zinc finger O ein ZNF468 isoform 2 F473 NM OO1 OO6656 Zinc finger O ein 473 F482 NM OO6626 Zinc finger O ein 482 F483 NM OO1007169 Zinc finger O ein 483 isoform b F490 NM 020714 Zinc finger O ein 490 F498 NM 145115 Zinc finger O ein 498 F500 NM 021646 Zinc finger O ein 500 F502 NM 03.3210 Zinc finger O ein 502 F510 NM O14930 Zinc finger O ein 510 F512 NM 032434 Zinc finger O ein 512 FS14 NM 032788 Zinc finger O ein 514 F518 NM 014803 Zinc finger O ein 518 F526 33444 Zinc finger O ein 526 F528 Zinc finger O ein 528 F532 Zinc finger O ein 532 F536 Zinc finger O ein 536 F542 Zinc finger O ein 542 FS46 Zinc finger O ein 546 F549 Zinc finger O ein 549 F551 Zinc finger O ein 551 F554 Zinc finger O ein 554 F556 Zinc finger O ein 556 F561 Zinc finger O ein 561 F562 Zinc finger O ein 562 F565 Zinc finger O ein 565 F566 Zinc finger O ein 566 F577 Zinc finger O en S77 F585A Zinc finger O ein 585A F587 Zinc finger O ein 587 F588 Zinc finger O ein 588 F595 Zinc finger O ein 595 F597 Zinc finger O ein 597 F599 Zinc finger O ein 599 isoform b F600 Zinc finger O ein 600 F62O Zinc finger O ein 620 F621 Zinc finger O ein 621 F623 Zinc finger O ein 623 F627 Zinc finger O ein 627 F651 NM Zinc finger O ein 651 F652 NM 014897 Zinc finger O ein 652 US 2009/0163434 A1 Jun. 25, 2009 59

TABLE 3-continued Predicted target genes of hsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description ZNF655 NM 001009956 Zinc finger protein 655 isoform e ZNF662 NM 2074.04 Zinc finger protein 662 ZNF66S NM O24733 Zinc finger protein 665 ZNF667 NM 022103 Zinc finger protein 667 ZNF669 NM 024804 Zinc finger protein 669 ZNF671 NM O24833 Zinc finger protein 671 ZNF68O NM 178558 Zinc finger protein 680 ZNF684 NM 152373 Zinc finger protein 684 ZNF69 NM O21915 Zinc finger protein 69 (Coss) ZNF696 NM O3O895 Zinc finger protein 696 ZNF70 NM 021916 Zinc finger protein 70 ZNF701 NM 018260 Zinc finger protein 701 ZNF702 NM 024924 Zinc finger protein 702 ZNF704 NM OO1033723 Zinc finger protein 704 ZNF708 NM 021269 Zinc finger protein 15-like 1 (KOX8) ZNF71 NM 021216 Zinc finger protein 71 ZNF721 NM 133474 Zinc finger protein 721 ZNF81 NM 007137 Zinc finger protein 81 (HFZ20) ZNFN1A4 NM O22465 Zinc finger protein, Subfamily 1A, 4 ZNFX1 NM 021035 Zinc finger, NFX1-type containing 1 ZSWIM3 NM 080752 Zinc finger, SWIM domain containing 3 ZSWIM4 NM 023072 Zinc finger, SWIM domain containing 4 ZXDB NM 007157 Zinc finger, X-linked, duplicated B ZYG11A NM OO1004339 hypothetical protein LOC440590 ZYG11B NM O24646 hypothetical protein LOC79699 ZZEF1 NM O15113 Zinc finger, ZZ type with EF hand domain 1 ZZZ3 NM O15534 Zinc finger, ZZ domain containing 3

TABLE 4 hsa-miR-20a targets that exhibited altered mRNA expression levels in human cancer cells after transfection with pre-miRhsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description ABCA1 NM OO55O2 ATP-binding cassette, sub-family A member 1 ANTXR1 NM 018153 tumor endothelial marker 8 isoform 3 precursor ARTS-1 NM 016442 type 1 tumor necrosis factor receptor shedding ATP6VOE NM OO3945 ATPase, H+ transporting, lysosomal, VO subunit ATP9A NM OO6045 ATPase, Class II, type 9A BICD2 NM OO10O3800 bicaudal Dhomolog 2 isoform 1 BTG3 NM OO6806 B-cell translocation gene 3 BTN3A2 NM 007047 butyrophilin, Subfamily 3, member A2 precursor C19orf2 NM 003796 RPB5-mediating protein isoform a C21orf25 NM 199050 hypothetical protein LOC25966 C6orf120 NM 001029863 hypothetical protein LOC387263 CCND1 NM O53056 cyclin D1 CDC37L1 NM 017913 cell division cycle 37 homolog (S. CLIC4 NM O13943 chloride intracellular channel 4 COL4A1 NM OO1845 alpha 1 type IV collagen preproprotein COL4A2 NM OO1846 alpha 2 type IV collagen preproprotein CPM NM 0010055O2 carboxypeptidase M precursor CRIPT NM 014171 postsynaptic protein CRIPT CXCLS NM OO2994 chemokine (C-X-C motif) ligand 5 precursor DAZAP2 NM 014764 DAZ associated protein 2 DCBLD2 NM 080927 discoidin, CUB and LCCL domain containing 2 DDAH1 NM 012137 dimethylarginine dimethylaminohydrolase 1 DNATB6 NM OO5494 DnaJ (Hsp40) homolog, subfamily B, member 6 DNAJC15 NM 013238 DNAJ domain-containing EIF2C1 NM 012199 eukaryotic translation initiation factor 2C, 1 EIF2S1 NM 004.094 eukaryotic translation initiation factor 2, EREG NM OO1432 epiregulin precursor F2RL1 NM OO5242 coagulation factor II (thrombin) receptor-like 1 FAM18B NM 016078 hypothetical protein LOC51030 FJX1 NM O14344 four jointed box 1 US 2009/0163434 A1 Jun. 25, 2009 60

TABLE 4-continued hsa-miR-20a targets that exhibited altered mRNA expression levels in human cancer cells after transfection with pre-miRhsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description FLJ31568 NM 152509 hypothetical protein LOC150244 FTS NM 00101.2398 fused toes homolog FYCO1 NM O24513 FYVE and coiled-coil domain containing 1 FZD7 NM 003507 rizzled 7 GATA6 NM OO5257 GATA binding protein 6 GNS NM OO2O76 glucosamine (N-acetyl)-6-sulfatase precursor GOLPH2 NM O16548 golgi phosphoprotein 2 HCCS NM 005333 holocytochrome c synthase (cytochrome c HIC2 NM O15094 hypermethylated in cancer 2 HMGA2 NM 001015886 high mobility group AT-hook 2 isoform c HN1 NM OO1002032 hematological and neurological expressed 1 L11 NM 000641 interleukin 11 precursor L8 NM OOO584 interleukin 8 precursor KCNMA1 NM 001014797 arge conductance calcium-activated potassium KIAAO494 NM 014774 hypothetical protein LOC9813 KLF10 NM OO1032282 Kruppel-like factor 10 isoform b LEPROT NM 017526 eptin receptor gene-related protein LHFP NM OO5780 ipoma HMGIC fusion partner LIMK1 NM 002314 LIM domain kinase 1 isoform 1 LRRCS4 NM O15516 Tsukushi M6PR NM 002355 cation-dependent mannose-6-phosphate receptor MAP3K2 NM OO6609 mitogen-activated protein kinase kinase kinase MGC11332 NM 032718 hypothetical protein LOC84804 MICA NM OOO247 MHC class I chain-related gene A protein NAGK NM 017567 N-Acetylglucosamine kinase NPAS2 NM OO2518 neuronal PAS domain protein 2 NPTX1 NM OO2522 neuronal pentraxin I precursor NRP2 NM 018534 neuropilin 2 isoform 4 precursor NUPL1 NM OO10O8564 nucleoporin like 1 isoform b OSTM1 NM 014028 Osteopetrosis associated transmembrane protein PDCD4 NM O14456 programmed cell death 4 isoform 1 PELI2 NM 021255 pellino 2 PFKP NM OO2627 phosphofructokinase, platelet PLAU NM 002658 urokinase plasminogen activator preproprotein PLCB1 NM O15192 phosphoinositide-specific phospholipase C beta 1 PON2 NM OOO305 paraOXonase 2 isoform 1 PTHLH NM 198965 parathyroid hormone-like hormone isoform 1 QKI NM 206853 quaking homolog, KH domain RNA binding isoform RAB22A NM O2O673 RAS-related protein RAB-22A RHOC NM 175744 ras homolog gene family, member C RNH1 NM OO2939 ribonucleasef angiogenin inhibitor RRM2 NM 001034 ribonucleotide reductase M2 polypeptide SERPINE1 NM OOO602 plasminogen activator inhibitor-1 SESN1 NM O14454 sestrin 1 SGPL1 NM OO3901 sphingosine-1-phosphate lyase 1 SLC1A4 NM OO3O38 solute carrier family 1, member 4 SLC2A3 NM OO6931 Solute carrier family 2 (facilitated glucose SNAP23 NM 003825 synaptosomal-associated protein 23 isoform SPARC NM OO3118 Secreted protein, acidic, cysteine-rich SPFH2 NM 007175 SPFH domain family, member 2 isoform 1 STC NM OO3155 Stanniocalcin 1 precursor SYNE1 NM O15293 nesprin 1 isoform beta TBC1D2 NM 018421 TBC1 domain family, member 2 TGFBR2 NM 001024847 TGF-beta type II receptor isoform A precursor TNFRSF10B NM OO3842 tumor necrosis factor receptor Superfamily, TXLNA NM 175852 Taxillin UEV3 NM 018314 ubiquitin-conjugating enzyme E2-like US 2009/0163434 A1 Jun. 25, 2009 61

TABLE 4-continued hsa-miR-20a targets that exhibited altered mRNA expression levels in human cancer cells after transfection with pre-miRhsa-miR-20a. RefSeq, Gene Transcript ID Symbol (Pruitt et al., 2005) Description USP46 NM O22832 ubiquitin specific protease 46 WANGL1 NM 138959 vang-like 1 VLDLR NM 001018056 very low density lipoprotein receptor isoform b WNTSA NM OO3392 wingless-type MMTV integration site family, ZNF331 NM 018555 Zinc finger protein 331

0053 Certain embodiments of the invention include deter in Some kit embodiments. The control molecules can be used mining expression of one or more marker, gene, or nucleic to verify transfection efficiency and/or control for transfec acid segment representative of one or more genes, by using an tion-induced changes in cells. amplification assay, a hybridization assay, or protein assay, a 0055 Certain embodiments are directed to a kit for assess variety of which are well known to one of ordinary skill in the ment of a pathological condition or the risk of developing a art. In certain aspects, an amplification assay can be a quan pathological condition in a patient by nucleic acid profiling of titative amplification assay, such as quantitative RT-PCR or a sample comprising, in Suitable container means, two or the like. In still further aspects, a hybridization assay can more nucleic acid hybridization or amplification reagents. include array hybridization assays or Solution hybridization The kit can comprise reagents for labeling nucleic acids in a assays. The nucleic acids from a sample may be labeled from sample and/or nucleic acid hybridization reagents. The the sample and/or hybridizing the labeled nucleic acid to one hybridization reagents typically comprise hybridization or more nucleic acid probes. Nucleic acids, mRNA, and/or probes. Amplification reagents include, but are not limited to nucleic acid probes may be coupled to a Support. Such Sup amplification primers, reagents, and enzymes. ports are well known to those of ordinary skill in the art and 0056. In some embodiments of the invention, an expres include, but are not limited to glass, plastic, metal, or latex. In particular aspects of the invention, the Support can be planar sion profile is generated by steps that include: (a) labeling or in the form of a bead or other geometric shapes or configu nucleic acid in the sample; (b) hybridizing the nucleic acid to rations known in the art. Proteins are typically assayed by a number of probes, or amplifying a number of nucleic acids, immunoblotting, chromatography, or mass spectrometry or and (c) determining and/or quantitating nucleic acid hybrid other methods known to those of ordinary skill in the art. ization to the probes or detecting and quantitating amplifica 0054 The present invention also concerns kits containing tion products, wherein an expression profile is generated. See compositions of the invention or compositions to implement U.S. Provisional Patent Application 60/575,743 and the U.S. methods of the invention. In some embodiments, kits can be Provisional Patent Application 60/649.584, and U.S. patent used to evaluate one or more marker molecules, and/or application Ser. No. 1 1/141,707 and U.S. patent application express one or more miRNA. In certain embodiments, a kit Ser. No. 1 1/273,640, all of which are hereby incorporated by contains, contains at least or contains at most 1, 2, 3, 4, 5, 6, reference. 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 0057 Methods of the invention involve diagnosing and/or 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37,38, 39, 40, 41, assessing the prognosis of a patient based on an miRNA 42, 43,44, 45,46, 47, 48,49, 50, 51, 52,53,54, 55,56, 57,58, and/or a marker nucleic acid expression profile. In certain 59, 60, 61,100, 150, 200 or more probes, recombinant nucleic embodiments, the elevation or reduction in the level of acid, or synthetic nucleic acid molecules related to the mark expression of a particular gene or genetic pathway or set of ers to be assessed oran miRNA to be expressed or modulated, nucleic acids in a cell is correlated with a disease state or and may include any range or combination derivable therein. pathological condition compared to the expression level of Kits may comprise components, which may be individually the same in a normal or non-pathologic cell or tissue sample. packaged or placed in a container, such as a tube, bottle, vial, This correlation allows for diagnostic and/or prognostic Syringe, or other Suitable container means. Individual com methods to be carried out when the expression level of one or ponents may also be provided in a kit in concentrated more nucleic acid is measured in a biological sample being amounts; in some embodiments, a component is provided assessed and then compared to the expression level of a nor individually in the same concentration as it would be in a mal or non-pathologic cell or tissue sample. It is specifically Solution with other components. Concentrations of compo contemplated that expression profiles for patients, particu nents may be provided as 1x, 2x, 5x, 10x, or 20x or more. Kits larly those Suspected of having or having a propensity for a for using probes, synthetic nucleic acids, recombinant nucleic particular disease or condition Such as cancer, can be gener acids, or non-synthetic nucleic acids of the invention for ated by evaluating any of or sets of the miRNAs and/or therapeutic, prognostic, or diagnostic applications are nucleic acids discussed in this application. The expression included as part of the invention. Specifically contemplated profile that is generated from the patient will be one that are any such molecules corresponding to any miRNA provides information regarding the particular disease or con reported to influence biological activity or expression of one dition. In many embodiments, the profile is generated using or more marker gene or gene pathway described herein. In nucleic acid hybridization or amplification, (e.g., array certain aspects, negative and/or positive controls are included hybridization or RT-PCR). In certain aspects, an expression US 2009/0163434 A1 Jun. 25, 2009 62 profile can be used in conjunction with other diagnostic and/ nucleic acids isolated from the sample, and (d) hybridizing or prognostic tests, such as histology, protein profiles in the the labeled nucleic acids to one or more probes. Nucleic acids serum and/or cytogenetic assessment. of the invention include one or more nucleic acid comprising 0058 The methods can further comprise one or more of at least one segment having a sequence or complementary the steps including: (a) obtaining a sample from the patient, sequence of to a nucleic acid representative of one or more of (b) isolating nucleic acids from the sample, (c) labeling the genes or markers in Table 1, 3, 4, and/or 5.

TABLE 5 Tumor associated mRNAS altered by hsa-miR-20a having prognostic or therapeutic value for the treatment of various malignancies.

Gene Cellular Symbol Gene Title Process Cancer Type Reference ANG angiogenin angiogenesis BC, OC, M, PaC, UC, CeC (Barton et al., 1997: Montero et al., 1998; Hartmann et al., 1999; Miyake et al., 1999; Shimoyama et al., 1999; Bodner-Adler et al., 2001) CCND1 cyclin D1 Cell cycle MCL, BC, SCCHN, OepC, (Donnellan and Chetty, 1998) HCC, CRC, BldC, EC, OC, M, AC, GB, GC, PaC CCNG1 cyclin G1 Cell cycle OS, BC, PC (Skotzko et al., 1995; Reimer et al., 1999) CEBPD CEBPdelta transcription PC (Yang et al., 2001) EPHB2 EPH receptor Signa PC, GC, CRC, OC, G, BC (Huusko et al., 2004; Nakada et al., 2004: Wu et al., 2004a: Jubb et al., B2 transduction 2005: Davalos et al., 2007: Guo et al., 2006: Kokko et al., 2006: Wu et al., 2006b) EREG epiregulin Signa BldC, CRC, PaC, PC (Baba et al., 2000; Torring et al., 2000; Zhu et al., 2000; Thogersen et al., transduction 2001) ETS2 ETS-2 transcription CeC, PC, TC, CRC, ESCC (Simpson et al., 1997: Sementchenko et al., 1998; de Nigris et al., 2001; Ito et al., 2002; Liet al., 2003) FGFR3 FGF-R3 Signa BldC, CRC, CeC, MM (LHote and Knowles, 2005) transduction FGFR4 FGF-R4 Signa TC, BC, OC, PaC (Jaakkola et al., 1993: Shah et al., 2002; Ezzat et al., 2005) transduction FZD7 Frizzled-7 Signa OepC, GC, HCC (Tanaka et al., 1998; Kirikoshi et al., 2001; Merle et al., 2004) transduction D4 inhibitor of transcription BC, GC, L (Chan et al., 2003;Yu et al., 2005; de Candia et al., 2006) DNA binding 4 GFBP1 GFBP-1 Signa BC, CRC (Firth and Baxter, 2002) transduction L8 L-8 Signa BC, CRC, PaC, NSCLC, PC, (Akiba et al., 2001; Sparmann and Bar-Sagi, 2004) transduction HCC AK1 anus kinase 1 Signa PC (Rossi et al., 2005) transduction JUN c-Jun transcription HL, HCC (Eferl et al., 2003; Weiss and Bohmann, 2004) LHFP ipoma transcription Li (Petit et al., 1999) HMGIC fusion battler LIMK1 LIM kinase 1 cell motility, BC, PC (Yoshioka et al., 2003) invasion P8 P8 transcription BC, TC, PaC (Ree et al., 1999: Su et al., 2001; Ito et al., 2005) PDGFRL PDGFR-like Signal CRC, NSCLC, HCC, PC (Fujiwara et al., 1995; Komiya et al., 1997) transduction PLCB1 PLC-beta1 Signal AML (Lo Vasco et al., 2004) transduction RARRES1 RAR migration, CRC, PC (Zhang et al., 2004: Wu et al., 2006a) responder 1 invasion RHOC RhoG cell motility, SCCHN, OepC, CRC, M, PC (Bellovin et al., 2006; Faried et al., 2006; Kleer et al., 2006; Ruth et al., invasion 2006; Yao et al., 2006) SKP2 SKP-2 proteasomal PaC, OC, BC, MFS, GB, EC, (Kamata et al., 2005; Saigusa et al., 2005; Shibahara et al., 2005; degradation NSCLC, PC Takanami, 2005; Einama et al., 2006; Huang et al., 2006; Sui et al., 2006; Traub et al., 2006) TGFBR2 TGFbeta Signal BC, CRC (Markowitz, 2000; Lucke et al., 2001; Biswas et al., 2004) receptor type transduction II TNFRSF1 OB TRAIL-R2 Apoptosis NSCLC, SCCHN, GC, BC, (Adams et al., 2005) US 2009/0163434 A1 Jun. 25, 2009 63

TABLE 5-continued Tumor associated mRNAS altered by hsa-miR-20a having prognostic or therapeutic value for the treatment of various malignancies. Gene Cellular Symbol Gene Title Process Cancer Type Reference VTN vitronectin Cell adhesion CRC, G, OC, M, BC (Tomasini-Johansson et al., 1994; Carreiras et al., 1996; Lee et al., 1998; Carreiras et al., 1999: Uhm et al., 1999; Aaboe et al., 2003) WNTSA Wnt-5a Signal NSCLC, BC, M, GC, NB (Saitoh et al., 2002: Blanc et al., 2005; Huang et al., 2005; Leris transduction et al., 2005) Abbreviations: AC, astrocytoma; AML, acute myelogenous leukemia; BC, breast carcinoma; BldC., bladder carcinoma: CeC, cervical carcinoma; CRC, colorectal carcinoma; EC, endometrial carcinoma; ESCC, esophageal squamous cell carcinoma; G, glioma: GB, glioblastoma; GC, gastric carcinoma; HCC, hepatocellular carcinoma; HL, Hodgkin lymphoma; L. leukemia; Li, lipoma; M, melanoma; MCL, mantle cell lymphoma; MFS, myxofibrosarcoma; MM, multiple myeloma; NB, neuroblastoma; NHL, non-Hodgkin lymphoma; NSCLC, non-Small cell lung carcinoma; OC, ovarian carcinoma; OepC, oesophageal carcinoma; OS, Osteosarcoma; PaC, pancreatic carcinoma; PC, prostate carcinoma; SCCHN, Squamous cell carcinoma of the head and neck; TC, thyroid carcinoma; UC, urothelial carcinoma.

0059. It is contemplated that any method or composition tive fragments thereof, unless otherwise indicated. It is under described herein can be implemented with respect to any stood by those of skill in the art that a “gene family’ refers to other method or composition described herein and that dif a group of genes having the same coding sequence or miRNA ferent embodiments may be combined. It is specifically con coding sequence. Typically, miRNA members of a gene fam templated that any methods and compositions discussed ily are identified by a number following the initial designa tion. For example, miR-16-1 and miR-16-2 are members of herein with respect to miRNA molecules, miRNA, genes, and the miR-16 gene family and “mir-7 refers to miR-7-1, miR nucleic acids representative of genes may be implemented 7-2 and miR-7-3. Moreover, unless otherwise indicated, a with respect to synthetic nucleic acids. In some embodiments shorthand notation refers to related miRNAs (distinguished the synthetic nucleic acid is exposed to the proper conditions by a letter). Exceptions to this shorthand notations will be to allow it to become a processed or mature nucleic acid, Such otherwise identified. as a miRNA under physiological circumstances. The claims 0062. Other embodiments of the invention are discussed originally filed are contemplated to cover claims that are throughout this application. Any embodiment discussed with multiply dependent on any filed claim or combination of filed respect to one aspect of the invention applies to other aspects claims. of the invention as well and vice versa. The embodiments in the Example and Detailed Description section are understood 0060 Also, any embodiment of the invention involving to be embodiments of the invention that are applicable to all specific genes (including representative fragments there of), aspects of the invention. mRNA, or miRNAs by name is contemplated also to cover 0063. The terms “inhibiting,” “reducing,” or “prevention.” embodiments involving miRNAS whose sequences are at or any variation of these terms, when used in the claims and/or least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90,91, 92,93, 94, the specification includes any measurable decrease or com 95, 96, 97,98, 99% identical to the mature sequence of the plete inhibition to achieve a desired result. specified miRNA. 0064. The use of the word “a” or “an' when used in con 0061. It will be further understood that shorthand nota junction with the term “comprising in the claims and/or the tions are employed Such that a generic description of a gene or specification may mean “one but it is also consistent with marker thereof, or of an miRNA refers to any of its gene the meaning of"one or more.” “at least one.” and “one or more family members (distinguished by a number) or representa than one.” US 2009/0163434 A1 Jun. 25, 2009 64

0065. Throughout this application, the term “about is endogenous miRNAS in cells, as well those genes and asso used to indicate that a value includes the standard deviation of ciated pathways modulated by the endogenous miRNA. error for the device or method being employed to determine 0073. The present invention concerns, in some embodi the value. ments, short nucleic acid molecules that function as miRNAS 0.066. The use of the term “or” in the claims is used to or as inhibitors of miRNA in a cell. The term "short” refers to mean “and/or unless explicitly indicated to refer to alterna a length of a single polynucleotide that is 15, 16, 17, 18, 19. tives only or the alternatives are mutually exclusive, although 20, 21, 22, 23, 24, 25, 50, 100, or 150 nucleotides or fewer, the disclosure supports a definition that refers to only alter including all integers or ranges derivable there between. The natives and “and/or.” nucleic acid molecules are typically synthetic. The term "syn 0067. As used in this specification and claim(s), the words thetic' refers to a nucleic acid molecule that is not produced “comprising (and any form of comprising, Such as "com naturally in a cell. In certain aspects the chemical structure prise' and "comprises”), “having (and any form of having, deviates from a naturally-occurring nucleic acid molecule, such as “have and “has'), “including (and any form of such as an endogenous precursor miRNA or miRNA mol including, such as “includes” and “include’) or “containing ecule. While in some embodiments, nucleic acids of the (and any form of containing, Such as “contains' and “con invention do not have an entire sequence that is identical to a tain’) are inclusive or open-ended and do not exclude addi sequence of a naturally-occurring nucleic acid, Such mol tional, unrecited elements or method steps. ecules may encompass all or part of a naturally-occurring 0068. Other objects, features and advantages of the sequence. It is contemplated, however, that a synthetic present invention will become apparent from the following nucleic acid administered to a cell may subsequently be detailed description. It should be understood, however, that modified or altered in the cell such that its structure or the detailed description and the specific examples, while indi sequence is the same as non-synthetic or naturally occurring cating specific embodiments of the invention, are given by nucleic acid, such as a mature miRNA sequence. For way of illustration only, since various changes and modifica example, a synthetic nucleic acid may have a sequence that tions within the spirit and scope of the invention will become differs from the sequence of a precursor miRNA, but that apparent to those skilled in the art from this detailed descrip sequence may be altered once in a cell to be the same as an tion. endogenous, processed miRNA. The term "isolated' means that the nucleic acid molecules of the invention are initially DETAILED DESCRIPTION OF THE INVENTION separated from different (in terms of sequence or structure) and unwanted nucleic acid molecules such that a population 0069. The present invention is directed to compositions of isolated nucleic acids is at least about 90% homogenous, and methods relating to the identification and characteriza and may be at least about 95.96, 97.98.99, or 100% homog tion of genes and biological pathways related to these genes enous with respect to other polynucleotide molecules. In as represented by the expression of the identified genes, as many embodiments of the invention, a nucleic acid is isolated well as use of miRNAs related to such, for therapeutic, prog by virtue of it having been synthesized in vitro separate from nostic, and diagnostic applications, particularly those meth endogenous nucleic acids in a cell. It will be understood, ods and compositions related to assessing and/or identifying however, that isolated nucleic acids may be Subsequently pathological conditions directly or indirectly related to miR mixed or pooled together. In certain aspects, synthetic 20a expression or the aberrant expression thereof. miRNA of the invention are RNA or RNA analogs. miRNA 0070. In certain aspects, the invention is directed to meth inhibitors may be DNA or RNA, or analogs thereof. miRNA ods for the assessment, analysis, and/or therapy of a cell or and miRNA inhibitors of the invention are collectively Subject where certain genes have a reduced or increased referred to as “synthetic nucleic acids.” expression (relative to normal) as a result of an increased or 0074. In some embodiments, there is a miRNA or a syn decreased expression of any one or a combination of miR-20 thetic miRNA having a length of between 17 and 130 resi family members. In certain instances the expression profile dues. The present invention concerns miRNA or synthetic and/or response to miR-20 expression or inhibition may be miRNA molecules that are, are at least, or are at most 15, 16, indicative of a disease or pathological condition, e.g., cancer. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 0071 Prognostic assays featuring any one or combination 34, 35,36, 37,38, 39, 40, 41,42, 43,44, 45,46, 47, 48,49, 50, of the miRNAs listed or the markers listed (including nucleic 51, 52,53,54, 55,56, 57,58, 59, 60, 61, 62,63, 64, 65,66, 67, acids representative thereof) could be used to assess an 68, 69,70, 71,72, 73,74, 75,76, 77,78, 79,80, 81, 82, 83, 84, patient to determine what if any treatment regimen is justi 85, 86, 87, 88, 89,90,91, 92,93, 94, 95, 96, 97,98, 99, 100, fied. As with the diagnostic assays mentioned above, the 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, absolute values that define low expression will depend on the 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, platform used to measure the miRNA(s). The same methods 125, 126, 127, 128, 129, 130, 140, 145, 150, 160, 170, 180, described for the diagnostic assays could be used for prog 190, 200 or more residues in length, including any integer or nostic assays. any range there between. 0075. In certain embodiments, synthetic miRNA have (a) I. Therapeutic Methods an “miRNA region' whose sequence or binding region from 5' to 3' is identical to all or a segment of a mature miRNA 0072 Embodiments of the invention concern nucleic sequence, and (b) a “complementary region' whose sequence acids that perform the activities of or inhibit endogenous from 5' to 3' is between 60% and 100% complementary to the miRNAs when introduced into cells. In certain aspects, miRNA sequence. In certain embodiments, these synthetic nucleic acids are synthetic or non-synthetic miRNA. miRNA are also isolated, as defined above. The term “miRNA Sequence-specific miRNA inhibitors can be used to inhibit region” refers to a region on the synthetic miRNA that is at sequentially or in combination the activities of one or more least 75, 80, 85, 90, 95, or 100% identical, including all US 2009/0163434 A1 Jun. 25, 2009

integers there between, to the entire sequence of a mature, 0080 Additional embodiments concern a synthetic naturally occurring miRNA sequence. In certain embodi miRNA having one or more Sugar modifications in the first or ments, the miRNA region is or is at least 90,91, 92,93, 94, 95, last 1 to 6 residues of the complementary region (referred to 96, 97,98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, as the 'Sugar replacement design). In certain cases, there is 99.9 or 100% identical to the sequence of a naturally-occur one or more Sugar modifications in the first 1, 2, 3, 4, 5, 6 or ring miRNA. more residues of the complementary region, or any range 0076. The term “complementary region” refers to a region derivable therein. In additional cases, there is one or more ofa synthetic miRNA that is or is at least 60% complementary Sugar modifications in the last 1, 2, 3, 4, 5, 6 or more residues to the mature, naturally occurring miRNA sequence. The of the complementary region, or any range derivable therein, complementary region is or is at least 60, 61, 62, 63, 64, 65, have a sugar modification. It will be understood that the terms 66, 67,68, 69,70, 71, 72,73,74, 75,76, 77,78, 79,80, 81,82, “first and “last’ are with respect to the order of residues from 83, 84,85, 86, 87, 88, 89,90,91, 92,93, 94.95, 96, 97,98,99, the 5' end to the 3' end of the region. In particular embodi 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% ments, the sugar modification is a 2"O-Me 2 F. modification, complementary, or any range derivable therein. With single a 2H modification, a 2'amino modification, a 4'thioribose polynucleotide sequences, there may be a hairpin loop struc modification, or a phosphorothioate modification on the car ture as a result of chemical bonding between the miRNA boxy group linked to the carbon at position 6'. In further region and the complementary region. In other embodiments, embodiments, there is one or more Sugar modifications in the the complementary region is on a different nucleic acid mol first or last 2 to 4 residues of the complementary region or the ecule than the miRNA region, in which case the complemen first or last 4 to 6 residues of the complementary region. This tary region is on the complementary Strand and the miRNA design element can also be used with an miRNA inhibitor. region is on the active strand. Thus, an miRNA inhibitor can have this design element and/ 0077. In other embodiments of the invention, there are or a replacement group on the nucleotide at the 5' terminus, as synthetic nucleic acids that are miRNA inhibitors. An miRNA discussed above. inhibitor is between about 17 to 25 nucleotides in length and 0081. In other embodiments of the invention, there is a comprises a 5' to 3' sequence that is at least 90% complemen synthetic miRNA in which one or more nucleotides in the last tary to the 5' to 3' sequence of a mature miRNA. In certain 1 to 5 residues at the 3' end of the complementary region are embodiments, an miRNA inhibitor molecule is 17, 18, 19, 20, not complementary to the corresponding nucleotides of the 21, 22, 23, 24, or 25 nucleotides in length, or any range miRNA region (“noncomplementarity') (referred to as the derivable therein. Moreover, an miRNA inhibitor may have a “noncomplementarity design). The noncomplementarity sequence (from 5' to 3') that is or is at least 70, 75, 80, 85,90, may be in the last 1, 2, 3, 4, and/or 5 residues of the comple 91, 92,93, 94, 95, 96, 97,98, 99,99.1, 99.2, 99.3, 99.4, 99.5, mentary miRNA. In certain embodiments, there is non 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range complementarity with at least 2 nucleotides in the comple derivable therein, to the 5' to 3' sequence of a mature miRNA, mentary region. particularly a mature, naturally occurring miRNA. One of I0082 It is contemplated that synthetic miRNA of the skill in the art could use a portion of the miRNA sequence that invention have one or more of the replacement, Sugar modi is complementary to the sequence of a mature miRNA as the fication, or noncomplementarity designs. In certain cases, sequence for an miRNA inhibitor. Moreover, that portion of synthetic RNA molecules have two of them, while in others the nucleic acid sequence can be altered so that it is still these molecules have all three designs in place. comprises the appropriate percentage of complementarity to I0083. The miRNA region and the complementary region the sequence of a mature miRNA. may be on the same or separate polynucleotides. In cases in 0078. In some embodiments, of the invention, a synthetic which they are contained on or in the same polynucleotide, miRNA contains one or more design element(s). These the miRNA molecule will be considered a single polynucle design elements include, but are not limited to: (i) a replace otide. In embodiments in which the different regions are on ment group for the phosphate or hydroxyl of the nucleotide at separate polynucleotides, the synthetic miRNA will be con the 5' terminus of the complementary region; (ii) one or more sidered to be comprised of two polynucleotides. sugar modifications in the first or last 1 to 6 residues of the I0084. When the RNA molecule is a single polynucleotide, complementary region; or, (iii) noncomplementarity between there can be a linker region between the miRNA region and one or more nucleotides in the last 1 to 5 residues at the 3' end the complementary region. In some embodiments, the single of the complementary region and the corresponding nucle polynucleotide is capable of forming a hairpin loop structure otides of the miRNA region. A variety of design modifica as a result of bonding between the miRNA region and the tions are known in the art, see below. complementary region. The linker constitutes the hairpin 0079. In certain embodiments, a synthetic miRNA has a loop. It is contemplated that in Some embodiments, the linker nucleotide at its 5' end of the complementary region in which region is, is at least, or is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, the phosphate and/or hydroxyl group has been replaced with 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, another chemical group (referred to as the “replacement 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 residues in design). In some cases, the phosphate group is replaced, length, or any range derivable therein. In certain embodi while in others, the hydroxyl group has been replaced. In ments, the linker is between 3 and 30 residues (inclusive) in particular embodiments, the replacement group is biotin, an length. amine group, a lower alkylamine group, an aminohexyl phos I0085. In addition to having an miRNA region and a phate group, an acetyl group, 2"O-Me (2'oxygen-methyl), complementary region, there may be flanking sequences as DMTO (4,4'-dimethoxytrity1 with oxygen), fluoroscein, a well at either the 5' or 3' end of the region. In some embodi thiol, or acridine, though other replacement groups are well ments, there is is at least 1, 2,3,4,5,6,7,8,9, 10 nucleotides known to those of skill in the art and can be used as well. This or more, or any range derivable therein, flanking one or both design element can also be used with an miRNA inhibitor. sides of these regions. US 2009/0163434 A1 Jun. 25, 2009 66

I0086 Methods of the invention include reducing or elimi list of miRNAS using Markush group language may be articu nating activity of one or more miRNAS in a cell comprising lated without the Markush group language and a disjunctive introducing into a cell an miRNA inhibitor, or Supplying or article (i.e., or) instead, and vice versa. enhancing the activity of one or more miRNAs in a cell. The 0090. In some embodiments, there is a method for reduc present invention also concerns inducing certain cellular ing or inhibiting cell proliferation in a cell comprising intro characteristics by providing to a cell aparticular nucleic acid, Such as a specific synthetic miRNA molecule or a synthetic ducing into or providing to the cell an effective amount of (i) miRNA inhibitor molecule. However, in methods of the an miRNA inhibitor molecule or (ii) a synthetic or nonsyn invention, the miRNA molecule or miRNA inhibitor need not thetic miRNA molecule that corresponds to a miRNA be synthetic. They may have a sequence that is identical to a sequence. In certain embodiments the methods involves naturally occurring miRNA or they may not have any design introducing into the cell an effective amount of (i) a miRNA modifications. In certain embodiments, the miRNA molecule inhibitor molecule having a 5' to 3' sequence that is at least and/oran miRNA inhibitor are synthetic, as discussed above. 90% complementary to the 5' to 3' sequence of one or more 0087. The particular nucleic acid molecule provided to the mature miRNA. cell is understood to correspond to a particular miRNA in the 0091 Certain embodiments of the invention include meth cell, and thus, the miRNA in the cell is referred to as the ods of treating a pathologic condition, in particular cancer, “corresponding miRNA. In situations in which a named e.g., lung or liver cancer. In one aspect, the method comprises miRNA molecule is introduced into a cell, the corresponding contacting a target cell with one or more nucleic acid, Syn miRNA will be understood to be the induced or inhibited thetic miRNA, or miRNA comprising at least one nucleic acid miRNA or miRNA function. It is contemplated, however, that segment having all or a portion of a miRNA sequence. The the miRNA molecule introduced into a cell is not a mature segment may be 5, 6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, miRNA but is capable of becoming a mature miRNA under 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides or nucleotide the appropriate physiological conditions. In cases in which a analog, including all integers there between. An aspect of the particular corresponding miRNA is being inhibited by a invention includes the modulation of gene expression, miRNA inhibitor, the particular miRNA will be referred to as the targeted miRNA. It is contemplated that multiple corre miRNA expression or function or mRNA expression or func sponding miRNAs may be involved. In particular embodi tion within a target cell. Such as a cancer cell. ments, more than one miRNA molecule is introduced into a 0092 Typically, an endogenous gene, miRNA or mRNA is cell. Moreover, in other embodiments, more than one miRNA modulated in the cell. In particular embodiments, the nucleic inhibitoris introduced into a cell. Furthermore, a combination acid sequence comprises at least one segment that is at least of miRNA molecule(s) and miRNA inhibitor(s) may be intro 70, 75, 80, 85, 90, 95, or 100% identical in nucleic acid duced into a cell. The inventors contemplate that a combina sequence to one or more miRNA or gene sequence. Modula tion of miRNA may act at one or more points in cellular tion of the expression or processing of an endogenous gene, pathways of cells with aberrant phenotypes and that Such miRNA, or mRNA can be through modulation of the process combination may have increased efficacy on the target cell ing of a mRNA, such processing including transcription, while not adversely effecting normal cells. Thus, a combina transportation and/or translation with in a cell. Modulation tion of miRNA may have a minimal adverse effect on a may also be effected by the inhibition or enhancement of Subject or patient while Supplying a Sufficient therapeutic miRNA activity with a cell, tissue, or organ. Such processing effect, Such as amelioration of a condition, growth inhibition may affect the expression of an encoded product or the sta of a cell, death of a targeted cell, alteration of cell phenotype bility of the mRNA. In still other embodiments, a nucleic acid or physiology, slowing of cellular growth, sensitization to a sequence can comprise a modified nucleic acid sequence. In second therapy, sensitization to a particular therapy, and the certain aspects, one or more miRNA sequence may include or like. comprise a modified nucleobase or nucleic acid sequence. 0088 Methods include identifying a cell or patient in need 0093. It will be understood in methods of the invention of inducing those cellular characteristics. Also, it will be that a cell or other biological matter Such as an organism understood that an amount of a synthetic nucleic acid that is (including patients) can be provided an miRNA or miRNA provided to a cellor organism is an “effective amount,” which molecule corresponding to a particular miRNA by adminis refers to an amount needed (or a sufficient amount) to achieve tering to the cell or organism a nucleic acid molecule that a desired goal. Such as inducing a particular cellular charac functions as the corresponding miRNA once inside the cell. teristic(s). In certain embodiments of the methods include The form of the molecule provided to the cell may not be the providing or introducing to a cell a nucleic acid molecule form that acts an miRNA once inside the cell. Thus, it is corresponding to a mature miRNA in the cell in an amount contemplated that in some embodiments, a synthetic miRNA effective to achieve a desired physiological result. or a nonsynthetic miRNA is provided such that it becomes 0089 Moreover, methods can involve providing synthetic processed into a mature and active miRNA once it has access or nonsynthetic miRNA molecules. It is contemplated that in to the cell's miRNA processing machinery. In certain these embodiments, that the methods may or may not be embodiments, it is specifically contemplated that the miRNA limited to providing only one or more synthetic miRNA mol molecule provided is not a mature miRNA molecule but a ecules or only one or more nonsynthetic miRNA molecules. nucleic acid molecule that can be processed into the mature Thus, in certain embodiments, methods may involve provid miRNA once it is accessible to miRNA processing machin ing both synthetic and nonsynthetic miRNA molecules. In ery. The term “nonsynthetic' in the context of miRNA means this situation, a cell or cells are most likely provided a syn that the miRNA is not “synthetic.' as defined herein. Further thetic miRNA molecule corresponding to a particular miRNA more, it is contemplated that in embodiments of the invention and a nonsynthetic miRNA molecule corresponding to a dif that concern the use of synthetic miRNAs, the use of corre ferent miRNA. Furthermore, any method articulated using a sponding nonsynthetic miRNAS is also considered an aspect US 2009/0163434 A1 Jun. 25, 2009 67 of the invention, and vice versa. It will be understand that the embodiments, there is a method of treating cancer in a patient term “providing an agent is used to include “administering comprising administering to the patient the cancer therapeu the agent to a patient. tic and an effective amount of at least one miRNA molecule 0094. In certain embodiments, methods also include tar that improves the efficacy of the cancertherapeutic or protects geting an miRNA to modulate in a cell or organism. The term non-cancer cells. Cancer therapies also include a variety of “targeting an miRNA to modulate” means a nucleic acid of combination therapies with both chemical and radiation the invention will be employed so as to modulate the selected based treatments. Combination chemotherapies include but miRNA. In some embodiments the modulation is achieved are not limited to, for example, 5-fluorouracil, alemtuzumab, with a synthetic or non-synthetic miRNA that corresponds to amrubicin, bevacizumab, bleomycin, bortezomib, buSulfan, the targeted miRNA, which effectively provides the targeted camptothecin, capecitabine, carboplatin, cetuximab, miRNA to the cellor organism (positive modulation). In other chlorambucil, cisplatin (CDDP), COX-2 inhibitors (e.g., embodiments, the modulation is achieved with an miRNA celecoxib), cyclophosphamide, cytarabine, dactinomycin, inhibitor, which effectively inhibits the targeted miRNA in dasatinib, daunorubicin, dexamethasone, docetaxel, doxoru the cell or organism (negative modulation). bicin (adriamycin), EGFR inhibitors (gefitinib and cetux 0095. In some embodiments, the miRNA targeted to be imab), erlotinib, estrogen receptor binding agents, etoposide modulated is an miRNA that affects a disease, condition, or (VP16), everolimus, farnesyl-protein transferase inhibitors, pathway. In certain embodiments, the miRNA is targeted gefitinib, gemcitabine, gemtuzumab, ibritumomab, ifosfa because a treatment can be provided by negative modulation mide, imatinib mesylate, larotaxel, lapatinib, lonafarnib, of the targeted miRNA. In other embodiments, the miRNA is mechlorethamine, melphalan, methotrexate, mitomycin, targeted because a treatment can be provided by positive navelbine, nitroSurea, nocodazole, oxaliplatin, paclitaxel, pli modulation of the targeted miRNA or its targets. comycin, procarbazine, raloxifene, rituximab, sirolimus, Sor 0096. In certain methods of the invention, there is a further afenib, Sunitinib, tamoxifen, taxol, taxotere, temsirolimus, step of administering the selected miRNA modulator to a cell, tipifarnib, to situmomab, transplatinum, trastuzumab, Vin tissue, organ, or organism (collectively “biological matter”) blastin, Vincristin, or vinorelbine or any analog or derivative in need of treatment related to modulation of the targeted variant of the foregoing. miRNA or in need of the physiological or biological results (0099 Generally, inhibitors of miRNAs can be given to discussed herein (such as with respect to a particular cellular decrease the activity of an endogenous miRNA. For example, pathway or result like decrease in cell viability). Conse inhibitors of miRNA molecules that increase cell prolifera quently, in some methods of the invention there is a step of tion can be provided to cells to decrease cell proliferation. The identifying a patient in need of treatment that can be provided present invention contemplates these embodiments in the by the miRNA modulator(s). It is contemplated that an effec context of the different physiological effects observed with tive amount of an miRNA modulator can be administered in the different miRNA molecules and miRNA inhibitors dis Some embodiments. In particular embodiments, there is a closed herein. These include, but are not limited to, the fol therapeutic benefit conferred on the biological matter, where lowing physiological effects: increase and decreasing cell a “therapeutic benefit” refers to an improvement in the one or proliferation, increasing or decreasing apoptosis, increasing more conditions or symptoms associated with a disease or transformation, increasing or decreasing cell viability, acti condition or an improvement in the prognosis, duration, or Vating or inhibiting a kinase (e.g., Erk), activating/inducing or status with respect to the disease. It is contemplated that a inhibiting hTert, inhibit stimulation of growth promoting therapeutic benefit includes, but is not limited to, a decrease in pathway (e.g., Stat3 signaling), reduce or increase viable cell pain, a decrease in morbidity, a decrease in a symptom. For number, and increase or decrease number of cells at a particu example, with respect to cancer, it is contemplated that a lar phase of the cell cycle. Methods of the invention are therapeutic benefit can be inhibition of tumor growth, preven generally contemplated to include providing or introducing tion of metastasis, reduction in number of metastases, inhi one or more different nucleic acid molecules corresponding bition of cancer cell proliferation, induction of cell death in to one or more different miRNA molecules. It is contemplated cancer cells, inhibition of angiogenesis near cancer cells, that the following, at least the following, or at most the fol induction of apoptosis of cancer cells, reduction in pain, lowing number of different nucleic acid or miRNA molecules reduction in risk of recurrence, induction of chemo- or radi may be provided or introduced: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, osensitivity in cancer cells, prolongation of life, and/or delay 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, of death directly or indirectly related to cancer. 29, 30, 31, 32,33,34, 35,36, 37,38, 39, 40, 41,42, 43,44, 45, 0097. Furthermore, it is contemplated that the miRNA 46,47, 48,49, 50, 51, 52,53,54, 55,56, 57,58, 59, 60, 61, 62, compositions may be provided as part of a therapy to a 63,64, 65,66, 67,68, 69,70, 71,72, 73,74, 75,76, 77,78,79, patient, in conjunction with traditional therapies or preventa 80, 81, 82, 83, 84,85, 86, 87, 88, 89,90,91, 92,93, 94, 95, 96, tive agents. Moreover, it is contemplated that any method 97, 98, 99, 100, or any range derivable therein. This also discussed in the context of therapy may be applied preventa applies to the number of different miRNA molecules that can tively, particularly in a patient identified to be potentially in be provided or introduced into a cell. need of the therapy or at risk of the condition or disease for which a therapy is needed. II. Pharmaceutical Formulations and Delivery 0098. In addition, methods of the invention concern 0100 Methods of the present invention include the deliv employing one or more nucleic acids corresponding to an ery of an effective amount of a miRNA or an expression miRNA and a therapeutic drug. The nucleic acid can enhance construct encoding the same. An "effective amount of the the effect or efficacy of the drug, reduce any side effects or pharmaceutical composition, generally, is defined as that toxicity, modify its bioavailability, and/or decrease the dos amount sufficient to detectably and repeatedly to achieve the age or frequency needed. In certain embodiments, the thera stated desired result, for example, to ameliorate, reduce, peutic drug is a cancer therapeutic. Consequently, in some minimize or limit the extent of the disease or its symptoms. US 2009/0163434 A1 Jun. 25, 2009

Other more rigorous definitions may apply, including elimi 0.108 Treatments may include various “unit doses. A unit nation, eradication or cure of disease. dose is defined as containing a predetermined quantity of a 0101 A. Administration therapeutic composition(s). The quantity to be administered, and the particular route and formulation, are within the skill 0102. In certain embodiments, it is desired to kill cells, of those in the clinical arts. A unit dose need not be adminis inhibit cell growth, inhibit metastasis, decrease tumor or tis tered as a single injection but may comprise continuous infu Sue size, and/or reverse or reduce the malignant or disease sion over a set period of time. With respect to a viral compo phenotype of cells. The routes of administration will vary, nent of the present invention, a unit dose may conveniently be naturally, with the location and nature of the lesion or site to described interms of ug or mg of miRNA or miRNA mimetic. be targeted, and include, e.g., intradermal, Subcutaneous, Alternatively, the amount specified may be the amount regional, parenteral, intravenous, intramuscular, intranasal, administered as the average daily, average weekly, or average systemic, and oral administration and formulation. Direct monthly dose. injection, intratumoral injection, or injection into tumor vas 0109 miRNA can be administered to the patient in a dose culature is specifically contemplated for discrete, Solid, or doses of about or of at least about 0.5, 1, 5, 10, 15, 20, 25, accessible tumors, or other accessible target areas. Local, 30, 35, 40, 45,50, 60, 70, 80,90, 100, 110, 120, 130, 140,150, regional, or systemic administration also may be appropriate. 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, For tumors of>4 cm, the volume to be administered will be 280, 290, 300, 310,320, 330, 340,350,360, 370, 380,390, about 4-10 ml (preferably 10 ml), while for tumors of <4 cm, 400, 410, 420, 430, 440, 450, 460, 470, 480,490, 500, 510, a volume of about 1-3 ml will be used (preferably 3 ml). 520, 530, 540, 550,560,570,580,590, 600, 610, 620, 630, 0103 Multiple injections delivered as a single dose com 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, prise about 0.1 to about 0.5 ml volumes. Compositions of the 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, invention may be administered in multiple injections to a 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, tumor or a targeted site. In certain aspects, injections may be 1000 ug or mg, or more, or any range derivable therein. spaced at approximately 1 cm intervals. Alternatively, the amount specified may be the amount 0104. In the case of surgical intervention, the present administered as the average daily, average weekly, or average invention may be used preoperatively, to render an inoperable monthly dose, or it may be expressed in terms of mg/kg, tumor Subject to resection. Alternatively, the present inven where kg refers to the weight of the patient and the mg is tion may be used at the time of Surgery, and/or thereafter, to specified above. In other embodiments, the amount specified treat residual or metastatic disease. For example, a resected is any number discussed above but expressed as mg/m (with tumor bed may be injected or perfused with a formulation respect to tumor size or patient Surface area). comprising a miRNA or combinations thereof. Administra 0110 B. Injectable Compositions and Formulations tion may be continued post-resection, for example, by leaving 0111. In some embodiments, the method for the delivery a catheter implanted at the site of the surgery. Periodic post of a miRNA or an expression construct encoding Such or Surgical treatment also is envisioned. Continuous perfusion combinations thereof is via systemic administration. How of an expression construct or a viral construct also is contem ever, the pharmaceutical compositions disclosed herein may plated. also be administered parenterally, Subcutaneously, directly, 0105 Continuous administration also may be applied intratracheally, intravenously, intradermally, intramuscu where appropriate, for example, where a tumor or other larly, or even intraperitoneally as described in U.S. Pat. Nos. undesired affected area is excised and the tumor bed or tar 5,543,158; 5,641,515 and 5,399,363 (each specifically incor geted site is treated to eliminate residual, microscopic dis porated herein by reference in its entirety). ease. Delivery via Syringe or catherization is contemplated. 0112 Injection of nucleic acids may be delivered by Such continuous perfusion may take place for a period from Syringe or any other method used for injection of a solution, about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to as long as the nucleic acid and any associated components can about 12-24 hours, to about 1-2 days, to about 1-2 wk or pass through the particular gauge of needle required for injec longer following the initiation of treatment. Generally, the tion. A syringe system has also been described for use in gene dose of the therapeutic composition via continuous perfusion therapy that permits multiple injections of predetermined will be equivalent to that given by a single or multiple injec quantities of a solution precisely at any depth (U.S. Pat. No. tions, adjusted over a period of time during which the perfu 5,846,225). sion occurs. 0113 Solutions of the active compounds as free base or 0106 Treatment regimens may vary as well and often pharmacologically acceptable salts may be prepared in water depend on tumor type, tumor location, immune condition, Suitably mixed with a surfactant, Such as hydroxypropylcel target site, disease progression, and health and age of the lulose. Dispersions may also be prepared in glycerol, liquid patient. Certain tumor types will require more aggressive polyethylene glycols, mixtures thereof, and in oils. Under treatment. The clinician will be best suited to make such ordinary conditions of storage and use, these preparations decisions based on the known efficacy and toxicity (if any) of contain a preservative to prevent the growth of microorgan the therapeutic formulations. isms. The pharmaceutical forms suitable for injectable use 0107. In certain embodiments, the tumor or affected area include sterile aqueous solutions or dispersions and sterile being treated may not, at least initially, be resectable. Treat powders for the extemporaneous preparation of sterile inject ments with compositions of the invention may increase the able solutions or dispersions (U.S. Pat. No. 5,466,468, spe resectability of the tumor due to shrinkage at the margins or cifically incorporated herein by reference in its entirety). In by elimination of certain particularly invasive portions. Fol all cases the form must be sterile and must be fluid to the lowing treatments, resection may be possible. Additional extent that easy Syringability exists. It must be stable under treatments Subsequent to resection may serve to eliminate the conditions of manufacture and storage and must be pre microscopic residual disease at the tumor or targeted site. served against the contaminating action of microorganisms, US 2009/0163434 A1 Jun. 25, 2009 69

Such as bacteria and fungi. The carrier can be a solvent or Suitable regimes for initial administration and Subsequent dispersion medium containing, for example, water, ethanol, administration are also variable, but are typified by an initial polyol (e.g., glycerol, propylene glycol, and liquid polyeth administration followed by other administrations. Such ylene glycol, and the like), Suitable mixtures thereof, and/or administration may be systemic, as a single dose, continuous vegetable oils. Proper fluidity may be maintained, for over a period of time spanning 10, 20, 30, 40, 50, 60 minutes, example, by the use of a coating, such as lecithin, by the and/or 1, 2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, maintenance of the required particle size in the case of dis 19, 20, 21, 22, 23, 24 or more hours, and/or 1, 2, 3, 4, 5, 6, 7, persion and by the use of surfactants. The prevention of the days or more. Moreover, administration may be through a action of microorganisms can be brought about by various time release or Sustained release mechanism, implemented by antibacterial and antifungal agents, for example, parabens, formulation and/or mode of administration. chlorobutanol, phenol, Sorbic acid, thimerosal, and the like. 0119 Various methods for nucleic acid delivery are In many cases, it will be preferable to include isotonic agents, described, for example in Sambrook et al., 1989, Molecular for example, Sugars or sodium chloride. Prolonged absorp Cloning: A Laboratory Manual, Cold Spring Harbor Labora tion of the injectable compositions can be brought about by tory, New York; and Ausubel et al., 1994, Current Protocols in the use in the compositions of agents delaying absorption, for Molecular Biology, John Wiley & Sons, New York. Such example, aluminum monostearate and gelatin. nucleic acid delivery systems comprise the desired nucleic 0114. In certain formulations, a water-based formulation acid, by way of example and not by limitation, in either is employed while in others, it may be lipid-based. In particu “naked’ form as a “naked nucleic acid, or formulated in a lar embodiments of the invention, a composition comprising vehicle suitable for delivery, such as in a complex with a a tumor Suppressor protein or a nucleic acid encoding the cationic molecule or a liposome forming lipid, or as a com same is in a water-based formulation. In other embodiments, ponent of a vector, or a component of a pharmaceutical com the formulation is lipid based. position. The nucleic acid delivery system can be provided to 0115 For parenteral administration in an aqueous solu the cell either directly, such as by contacting it with the cell, tion, for example, the solution should be suitably buffered if or indirectly, such as through the action of any biological necessary and the liquid diluent first rendered isotonic with process. By way of example, and not by limitation, the nucleic Sufficient Saline or glucose. These particular aqueous solu acid delivery system can be provided to the cell by endocy tions are especially suitable for intravenous, intramuscular, tosis; receptor targeting; coupling with native or synthetic cell Subcutaneous, intratumoral, intralesional, and intraperitoneal membrane fragments; physical means such as electropora administration. In this connection, sterile aqueous media tion; combining the nucleic acid delivery system with a poly which can be employed will be known to those of skill in the meric carrier, Such as a controlled release film or nanoparticle art in light of the present disclosure. For example, one dosage or microparticle or biocompatible molecules or biodegrad may be dissolved in 1 ml of isotonic NaCl solution and either able molecules; with vector. The nucleic acid delivery system added to 1000 ml of hypodermoclysis fluid or injected at the can be injected into a tissue or fluid Surrounding the cell, or proposed site of infusion, (see for example, "Remington's administered by diffusion of the nucleic acid delivery system Pharmaceutical Sciences' 15th Edition, pages 1035-1038 across the cell membrane, or by any active or passive trans and 1570-1580). Some variation in dosage will necessarily port mechanism across the cell membrane. Additionally, the occur depending on the condition of the Subject being treated. nucleic acid delivery system can be provided to the cell using The person responsible for administration will, in any event, techniques such as antibody-related targeting and antibody determine the appropriate dose for the individual subject. mediated immobilization of a viral vector. Moreover, for human administration, preparations should 0120 C. Combination Treatments meet Sterility, pyrogenicity, general safety and purity stan I0121. In certain embodiments, the compositions and dards as required by FDA Office of Biologics standards. methods of the present invention involve a miRNA, or expres 0116. As used herein, a “carrier' includes any and all sion construct encoding such. These miRNA composition can Solvents, dispersion media, vehicles, coatings, diluents, anti be used in combination with a second therapy to enhance the bacterial and antifungal agents, isotonic and absorption effect of the miRNA therapy, or increase the therapeutic effect delaying agents, buffers, carrier Solutions, Suspensions, col of another therapy being employed. These compositions loids, and the like. The use of Such media and agents for would be provided in a combined amount effective to achieve pharmaceutical active Substances is well known in the art. the desired effect, such as the killing of a cancer cell and/or Except insofar as any conventional media or agent is incom the inhibition of cellular hyperproliferation. This process may patible with the active ingredient, its use in the therapeutic involve contacting the cells with the miRNA or second compositions is contemplated. Supplementary active ingre therapy at the same or different time. This may beachieved by dients can also be incorporated into the compositions. contacting the cell with one or more compositions or phar 0117 The phrase “pharmaceutically acceptable' refers to macological formulation that includes or more of the agents, molecular entities and compositions that do not produce an or by contacting the cell with two or more distinct composi allergic or similar untoward reaction when administered to a tions or formulations, wherein one composition provides (1) human. miRNA; and/or (2) a second therapy. A second composition 0118. The nucleic acid(s) are administered in a manner or method may be administered that includes a chemotherapy, compatible with the dosage formulation, and in Such amount radiotherapy, Surgical therapy, immunotherapy or gene as will be therapeutically effective. The quantity to be admin therapy. istered depends on the Subject to be treated, including, e.g., I0122. It is contemplated that one may provide a patient the aggressiveness of the disease or cancer, the size of any with the miRNA therapy and the second therapy within about tumor(s) or lesions, the previous or other courses of treat 12-24 h of each other and, more preferably, within about 6-12 ment. Precise amounts of active ingredient required to be h of each other. In some situations, it may be desirable to administered depend on the judgment of the practitioner. extend the time period for treatment significantly, however, US 2009/0163434 A1 Jun. 25, 2009 70 where several days (2,3,4, 5, 6 or 7) to several weeks (1,2,3, aberrations by affecting nucleic acid synthesis. Most chemo 4, 5, 6, 7 or 8) lapse between the respective administrations. therapeutic agents fall into the following categories: alkylat 0123. In certain embodiments, a course of treatment will ing agents, antimetabolites, antitumor antibiotics, mitotic last 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, inhibitors, and nitrosoureas. 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34,35, 36, I0129. a. Alkylating Agents 37,38,39, 40, 41, 42, 43,44, 45,46, 47, 48,49, 50, 51, 52,53, 0.130 Alkylating agents are drugs that directly interact 54, 55,56, 57,58, 59, 60, 61, 62,63, 64, 65,66, 67,68, 69,70, with genomic DNA to prevent the cancer cell from prolifer 71, 72,73,74, 75,76, 77,78, 79,80, 81, 82,83, 84,85, 86, 87, ating. This category of chemotherapeutic drugs represents 88, 89,90 days or more. It is contemplated that one agent may agents that affect all phases of the cell cycle, that is, they are be given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, not phase-specific. Alkylating agents can be implemented to 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, treat chronic leukemia, non-Hodgkin’s lymphoma, 33,34,35,36, 37,38, 39, 40, 41, 42, 43,44, 45,46, 47, 48,49, Hodgkin's disease, multiple myeloma, and particular cancers 50, 51, 52,53,54, 55,56, 57,58, 59, 60, 61, 62,63, 64, 65,66, of the breast, lung, and ovary. They include: buSulfan, 67,68, 69,70, 71,72, 73,74, 75,76, 77,78, 79,80, 81, 82,83, chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacar 84, 85,86, 87.88, 89, and/or 90, any combination thereof, and bazine, ifosfamide, mechlorethamine (mustargen), and mel another agent is given on day 1, 2, 3, 4, 5, 6,7,8,9, 10, 11, 12. phalan. Troglitazaone can be used to treat cancer in combi 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, nation with any one or more of these alkylating agents. 30, 31, 32,33, 34,35, 36, 37,38, 39, 40, 41,42, 43,44, 45,46, 0131 b. Antimetabolites 47, 48,49, 50, 51, 52,53,54, 55,56, 57,58, 59, 60, 61, 62,63, I0132 Antimetabolites disrupt DNA and RNA synthesis. 64, 65,66, 67,68, 69,70,71, 72,73,74, 75,76, 77,78, 79,80, Unlike alkylating agents, they specifically influence the cell 81, 82, 83, 84,85, 86, 87, 88, 89, and/or 90, or any combina cycle during S phase. They have been used to combat chronic tion thereof. Within a single day (24-hour period), the patient leukemias in addition to tumors of breast, ovary and the may be given one or multiple administrations of the agent(s). gastrointestinal tract. Antimetabolites include 5-fluorouracil Moreover, after a course of treatment, it is contemplated that (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and there is a period of time at which no treatment is administered. methotrexate. This time period may last 1, 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 0.133 5-Fluorouracil (5-FU) has the chemical name of 3, 4, 5 weeks, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months 5-fluoro-2,4(1H.3H)-pyrimidinedione. Its mechanism of or more, depending on the condition of the patient, such as action is thought to be by blocking the methylation reaction of their prognosis, strength, health, etc. deoxyuridylic acid to thymidylic acid. Thus, 5-FU interferes 0.124 Various combinations may be employed, for with the synthesis of deoxyribonucleic acid (DNA) and to a example miRNA therapy is “A” and a second therapy is “B”: lesser extent inhibits the formation of ribonucleic acid

ABA B.A.B. B.B.A AAB ABB BAA AB,BB BABB B.B.B.A. B.B.AB AABB ABAB ABBA BBAA BAB, A BAAB AAAB BAAA ABAA AABA

0.125 Administration of any compound or therapy of the (RNA). Since DNA and RNA are essential for cell division present invention to a patient will follow general protocols for and proliferation, it is thought that the effect of 5-FU is to the administration of Such compounds, taking into account create a thymidine deficiency leading to cell death. Thus, the the toxicity, if any, of the vector or any protein or other agent. effect of 5-FU is found in cells that rapidly divide, a charac Therefore, in Some embodiments there is a step of monitoring teristic of metastatic cancers. toxicity that is attributable to combination therapy. It is expected that the treatment cycles would be repeated as nec 0.134 c. Antitumor Antibiotics essary. It also is contemplated that various standard therapies, 0.135 Antitumor antibiotics have both antimicrobial and as well as Surgical intervention, may be applied in combina cytotoxic activity. These drugs also interfere with DNA by tion with the described therapy. chemically inhibiting enzymes and mitosis or altering cellu 0126. In specific aspects, it is contemplated that a second lar membranes. These agents are not phase specific So they therapy. Such as chemotherapy, radiotherapy, immuno work in all phases of the cell cycle. Thus, they are widely used therapy, Surgical therapy or other genetherapy, is employed in for a variety of cancers. Examples of antitumor antibiotics combination with the miRNA therapy, as described herein. include bleomycin, dactinomycin, daunorubicin, doxorubi O127 1. Chemotherapy cin (Adriamycin), and idarubicin, Some of which are dis 0128. A wide variety of chemotherapeutic agents may be cussed in more detail below. Widely used in clinical setting used in accordance with the present invention. The term “che for the treatment of neoplasms, these compounds are admin motherapy” refers to the use of drugs to treat cancer. A “che istered through bolus injections intravenously at doses rang motherapeutic agent' is used to connote a compound or com ing from 25-75 mg/m at 21 day intervals for adriamycin, to position that is administered in the treatment of cancer. These 35-100 mg/m for etoposide intravenously or orally. agents or drugs are categorized by their mode of activity 0.136 d. Mitotic Inhibitors within a cell, for example, whether and at what stage they 0.137 Mitotic inhibitors include plant alkaloids and other affect the cell cycle. Alternatively, an agent may be charac natural agents that can inhibit either protein synthesis terized based on its ability to directly cross-link DNA, to required for cell division or mitosis. They operate during a intercalate into DNA, or to induce chromosomal and mitotic specific phase during the cell cycle. Mitotic inhibitors com US 2009/0163434 A1 Jun. 25, 2009

prise docetaxel, etoposide (VP16), paclitaxel, taxol, taxotere, cholera toxin, pertussis toxin, etc.) and serve merely as a vinblastine, Vincristine, and vinorelbine. targeting agent. Alternatively, the effector may be a lympho 0138 e. Nitrosureas cyte carrying a surface molecule that interacts, either directly 0139 Nitrosureas, like alkylating agents, inhibit DNA or indirectly, with a tumor cell target. Various effector cells repair proteins. They are used to treat non-Hodgkin’s lym include cytotoxic T cells and NK cells. The combination of phomas, multiple myeloma, malignant melanoma, in addi therapeutic modalities, i.e., direct cytotoxic activity and inhi tion to brain tumors. Examples include carmustine and bition or reduction of ErbB2 would provide therapeutic ben lomustine. efit in the treatment of ErbB2 overexpressing cancers. 0140 2. Radiotherapy 0146 In one aspect of immunotherapy, the tumor or dis 0141 Radiotherapy, also called radiation therapy, is the ease cell must bear Some marker that is amenable to targeting, treatment of cancer and other diseases with ionizing radia i.e., is not present on the majority of other cells. Many tumor tion. Ionizing radiation deposits energy that injures or markers exist and any of these may be suitable for targeting in destroys cells in the area being treated by damaging their the context of the present invention. Common tumor markers genetic material, making it impossible for these cells to con include carcinoembryonic antigen, prostate specific antigen, tinue to grow. Although radiation damages both cancer cells urinary tumor associated antigen, fetal antigen, tyrosinase and normal cells, the latter are able to repair themselves and (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, function properly. Radiotherapy may be used to treat local Much3, PLAP, estrogen receptor, laminin receptor, erbB and ized solid tumors, such as cancers of the skin, tongue, larynx, p155. An alternative aspect of immunotherapy is to combine brain, breast, or cervix. It can also be used to treat leukemia anticancer effects with immune stimulatory effects. Immune and lymphoma (cancers of the blood-forming cells and lym stimulating molecules also exist including: cytokines Such as phatic system, respectively). IL-2, IL-4, IL-12, GM-CSF, gamma-IFN. chemokines such 0142 Radiation therapy used according to the present as MIP-1, MCP-1, IL-8 and growth factors such as FLT3 invention may include, but is not limited to, the use of Y-rays, ligand. Combining immune stimulating molecules, either as X-rays, and/or the directed delivery of radioisotopes to tumor proteins or using gene delivery in combination with a tumor cells. Other forms of DNA damaging factors are also contem Suppressor Such as MDA-7 has been shown to enhance anti plated such as microwaves, proton beam irradiation (U.S. Pat. tumor effects (Ju et al., 2000). Moreover, antibodies against Nos. 5,760,395 and 4,870,287) and UV-irradiation. It is most any of these compounds can be used to target the anti-cancer likely that all of these factors effect a broad range of damage agents discussed herein. on DNA, on the precursors of DNA, on the replication and 0147 Examples of immunotherapies currently under repair of DNA, and on the assembly and maintenance of investigation or in use are immune adjuvants e.g., Mycobac chromosomes. Dosage ranges for X-rays range from daily terium bovis, Plasmodium falciparum, dinitrochlorobenzene doses of 50 to 200 roentgens for prolonged periods of time (3 and aromatic compounds (U.S. Pat. Nos. 5,801,005 and to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage 5,739,169: Hui and Hashimoto, 1998; Christodoulides et al., ranges for radioisotopes vary widely, and depend on the half 1998), cytokine therapy e.g., interferons C, B and Y: IL-1, life of the isotope, the strength and type of radiation emitted, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., and the uptake by the neoplastic cells. Radiotherapy may 1998: Hellstrand et al., 1998) gene therapy e.g., TNF, IL-1, comprise the use of radiolabeled antibodies to deliver doses IL-2, p53 (Qinet al., 1998; Austin-Ward and Villaseca, 1998: of radiation directly to the cancer site (radioimmunotherapy). U.S. Pat. Nos. 5,830,880 and 5,846,945) and monoclonal Once injected into the body, the antibodies actively seek out antibodies e.g., anti- GM2, anti-HER-2, anti the cancer cells, which are destroyed by the cell-killing (cyto p185; Pietras et al., 1998: Hanibuchietal., 1998: U.S. Pat. No. toxic) action of the radiation. This approach can minimize the 5,824.311). Herceptin (trastuzumab) is a chimeric (mouse risk of radiation damage to healthy cells. human) monoclonal antibody that blocks the HER2-neu 0143 Stereotactic radio-surgery (gamma knife) for brain receptor. It possesses anti-tumor activity and has been and other tumors does not use a knife, but very precisely approved for use in the treatment of malignant tumors targeted beams of gamma radiotherapy from hundreds of (Dillman, 1999). Table 6 is a non-limiting list of several different angles. Only one session of radiotherapy, taking known anti-cancer immunotherapeutic agents and their tar about four to five hours, is needed. For this treatment a spe gets. It is contemplated that one or more of these therapies cially made metal frame is attached to the head. Then, several may be employed with the miRNA therapies described scans and X-rays are carried out to find the precise area where herein. the treatment is needed. During the radiotherapy for brain tumors, the patient lies with their head in a large helmet, TABLE 6 which has hundreds of holes in it to allow the radiotherapy beams through. Related approaches permit positioning for Generic Name Target the treatment of tumors in other areas of the body. Cetuximab EGFR 0144) 3. Immunotherapy Panitumumab EGFR Trastuzumab erbB2 receptor 0145. In the context of cancer treatment, immunothera Bevacizumab VEGF peutics, generally, rely on the use of immune effector cells Alemtuzumab CD52 and molecules to target and destroy cancer cells. Trastuzumab gemtuzumab ozogamicin CD33 (HerceptinTM) is such an example. The immune effector may Rituximab CD2O be, for example, an antibody specific for Some marker on the Tositumomab CD2O Matuzumab EGFR Surface of a tumor cell. The antibody alone may serve as an ibritumomab tiuxetan CD2O effector of therapy or it may recruit other cells to actually Tositumomab CD2O effect cell killing. The antibody also may be conjugated to a HuPAM4 MUC1 drug or toxin (chemotherapeutic, radionuclide, ricin. A chain, US 2009/0163434 A1 Jun. 25, 2009 72

its CDK4, and thus may regulate Rb phosphorylation (Ser TABLE 6-continued rano et al., 1993: Serrano et al., 1995). Since the p16INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this Generic Name Target gene may increase the activity of CDK4, resulting in hyper MORAb-009 Mesothelin phosphorylation of the Rb protein. p16 also is known to G2SO carbonic anhydrase IX regulate the function of CDK6. mAb 8H9 8H9 antigen M195 CD33 0153 p16INK4 belongs to a newly described class of pilimumab CTLA4 CDK-inhibitory proteins that also includes p16B, p19. HuLuc63 CS1 p21WAF1, and p27KIP1. The p16INK4 gene maps to 9p21, a Alemtuzumab CD53 chromosome region frequently deleted in many tumor types. Epratuzumab CD22 Homozygous deletions and mutations of the p16INK4 gene BC8 CD45 are frequent in human tumor cell lines. This evidence Sug HuJS91 Prostate specific membrane antigen gests that the p16INK4 gene is a tumor Suppressor gene. This Lexatumumab TRAIL receptor-2 interpretation has been challenged, however, by the observa Pertuzumab HER-2 receptor Mik-beta-1 IL-2R tion that the frequency of the p16INK4 gene alterations is RAV12 RAAG12 much lower in primary uncultured tumors than in cultured SGN-30 CD30 cell lines (Caldas et al., 1994; Cheng et al., 1994; Hussussian AME-133w CD2O et al., 1994; Kamb et al., 1994; Mori et al., 1994: Okamoto et HeFi-1 CD30 BMS-663S13 CD137 al., 1994: Nobori et al., 1995: Orlow et al., 1994; Arap et al., Volociximab anti-C531 integrin 1995). Restoration of wild-type p16INK4 function by trans GC1008 TGFB fection with a plasmid expression vector reduced colony for HCD122 CD40 mation by some human cancer cell lines (Okamoto, 1994: Siplizumab CD2 MORAb-003 Folate receptor alpha Arap, 1995). CNTO 328 IL-6 0154) Other genes that may be employed according to the MDX-060 CD30 present invention include Rb, APC, DCC, NF-1, NF-2, WT-1, Ofatumumab CD2O MEN-I, MEN-II, Zac1, p73, VHL, MMAC1/PTEN, DBCCR SGN-33 CD33 1. FCC, rsk-3, p27, p27/p16 fusions, p21/p27 fusions, anti thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, 0148. A number of different approaches for passive immu myc, neu, raf, erb, fms, trk, ret, gsp. hist, abl, E1A, p300, genes notherapy of cancer exist. They may be broadly categorized involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, into the following: injection of antibodies alone; injection of BAI-1, GDAIF, or their receptors) and MCC. antibodies coupled to toxins or chemotherapeutic agents; (O155 5. Surgery injection of antibodies coupled to radioactive isotopes; injec 0156 Approximately 60% of persons with cancer will tion of anti-idiotype antibodies; and finally, purging of tumor undergo Surgery of some type, which includes preventative, cells in bone marrow. diagnostic or staging, curative and palliative Surgery. Cura 0149 4. Gene Therapy tive Surgery is a cancer treatment that may be used in con 0150. In yet another embodiment, a combination treat junction with other therapies, such as the treatment of the ment involves gene therapy in which atherapeutic polynucle present invention, chemotherapy, radiotherapy, hormonal otide is administered before, after, or at the same time as one therapy, gene therapy, immunotherapy and/or alternative or more therapeutic miRNA. Delivery of a therapeutic therapies. polypeptide or encoding nucleic acid in conjunction with a 0157 Curative surgery includes resection in which all or miRNA may have a combined therapeutic effect on target part of cancerous tissue is physically removed, excised, and/ tissues. A variety of proteins are encompassed within the or destroyed. Tumor resection refers to physical removal of at invention, some of which are described below. Various genes least part of a tumor. In addition to tumor resection, treatment that may be targeted for gene therapy of some form in com by Surgery includes laser Surgery, cryoSurgery, electroSur bination with the present invention include, but are not lim gery, and microscopically controlled Surgery (Mohs Sur ited to inducers of cellular proliferation, inhibitors of cellular gery). It is further contemplated that the present invention proliferation, regulators of programmed cell death, cytokines may be used in conjunction with removal of Superficial can and other therapeutic nucleic acids or nucleic acid that encode cers, precancers, or incidental amounts of normal tissue. therapeutic proteins. 0158 Upon excision of part of all of cancerous cells, tis 0151. The tumor suppressor oncogenes function to inhibit Sue, or tumor, a cavity may be formed in the body. Treatment excessive cellular proliferation. The inactivation of these may be accomplished by perfusion, direct injection or local genes destroys their inhibitory activity, resulting in unregu application of the area with an additional anti-cancer therapy. lated proliferation. The tumor Suppressors (e.g., therapeutic Such treatment may be repeated, for example, every 1, 2, 3, 4, polypeptides) p53, FHIT, p16 and C-CAM can be employed. 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 0152. In addition to p53, another inhibitor of cellular pro 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may liferation is p16. The major transitions of the eukaryotic cell be of varying dosages as well. cycle are triggered by cyclin-dependent kinases, or CDKs. 0159. 6. Other Agents One CDK, cyclin-dependent kinase 4 (CDK4), regulates pro 0160. It is contemplated that other agents may be used in gression through the G1. The activity of this enzyme may be combination with the present invention to improve the thera to phosphorylate Rb at late G1. The activity of CDK4 is peutic efficacy of treatment. These additional agents include controlled by an activating subunit, D-type cyclin, and by an immunomodulatory agents, agents that affect the upregula inhibitory subunit, the p16INK4 has been biochemically tion of cell Surface receptors and GAP junctions, cytostatic characterized as a protein that specifically binds to and inhib and differentiation agents, inhibitors of cell adhesion, agents US 2009/0163434 A1 Jun. 25, 2009

that increase the sensitivity of the hyperproliferative cells to outside the body. Internal heat may involve a sterile probe, apoptotic inducers, or other biological agents. Immunomodu including thin, heated wires or hollow tubes filled with warm latory agents include tumor necrosis factor, interferon alpha, water, implanted microwave antennae, or radiofrequency beta, and gamma; IL-2 and other cytokines; F42K and other electrodes. cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, 0164. A patient's organ or a limb is heated for regional and other chemokines. It is further contemplated that the therapy, which is accomplished using devices that produce upregulation of cell Surface receptors or their ligands such as high energy, Such as magnets. Alternatively, some of the Fas/Fas ligand, DR4 or DR5/TRAIL (Apo-2 ligand) would patient's blood may be removed and heated before being potentiate the apoptotic inducing abilities of the present perfused into an area that will be internally heated. Whole invention by establishment of an autocrine or paracrine effect body heating may also be implemented in cases where cancer on hyperproliferative cells. Increases intercellular signaling has spread throughout the body. Warm-water blankets, hot by elevating the number of GAPjunctions would increase the wax, inductive coils, and thermal chambers may be used for anti-hyperproliferative effects on the neighboring hyperpro this purpose. liferative cell population. In other embodiments, cytostatic or 0.165 Hormonal therapy may also be used in conjunction differentiation agents can be used in combination with the with the present invention or in combination with any other present invention to improve the anti-hyperproliferative effi cancer therapy previously described. The use of hormones cacy of the treatments. Inhibitors of cell adhesion are con may be employed in the treatment of certain cancers such as templated to improve the efficacy of the present invention. breast, prostate, ovarian, or cervical cancer to lower the level Examples of cell adhesion inhibitors are focal adhesion or block the effects of certain hormones such as testosterone kinase (FAKs) inhibitors and Lovastatin. It is further contem or estrogen. This treatment is often used in combination with plated that other agents that increase the sensitivity of a hyper at least one other cancer therapy as a treatment option or to proliferative cell to apoptosis, such as the antibody c225, reduce the risk of metastases. could be used in combination with the present invention to 0166 This application incorporates U.S. application Ser. improve the treatment efficacy. No. 1 1/349,727 filed on Feb. 8, 2006 claiming priority to U.S. 0161 Apo2 ligand (Apo2L, also called TRAIL) is a mem Provisional Application Ser. No. 60/650,807 filed Feb. 8, ber of the tumor necrosis factor (TNF) cytokine family. 2005 herein by references in its entirety. TRAIL activates rapid apoptosis in many types of cancer cells, yet is not toxic to normal cells. TRAIL mRNA occurs in III. miRNA Molecules a wide variety of tissues. Most normal cells appear to be (0167 MicroRNA molecules (“miRNAs) are generally resistant to TRAIL's cytotoxic action, Suggesting the exist 21 to 22 nucleotides in length, though lengths of 19 and up to ence of mechanisms that can protect againstapoptosis induc 23 nucleotides have been reported. The miRNAs are each tion by TRAIL. The first receptor described for TRAIL, processed from a longerprecursor RNA molecule (“precursor called death receptor 4 (DR4), contains a cytoplasmic “death miRNA). Precursor miRNAs are transcribed from non-pro domain': DR4 transmits the apoptosis signal carried by tein-encoding genes. The precursor miRNAS have two TRAIL. Additional receptors have been identified that bind to regions of complementarity that enables them to form a stem TRAIL. One receptor, called DR5, contains a cytoplasmic loop- or fold-back-like structure, which is cleaved in animals death domain and signals apoptosis much like DR4. The DR4 by a ribonuclease III-like nuclease enzyme called Dicer. The and DR5 mRNAs are expressed in many normal tissues and processed miRNA is typically a portion of the stem. tumor cell lines. Recently, decoy receptors such as DcR1 and 0.168. The processed miRNA (also referred to as “mature DcR2 have been identified that prevent TRAIL from inducing miRNA) becomes part of a large complex to down-regulate apoptosis through DR4 and DR5. These decoy receptors thus a particular target gene or its gene product. Examples of represent a novel mechanism for regulating sensitivity to a animal miRNAs include those that imperfectly basepair with pro-apoptotic cytokine directly at the cell's surface. The pref the target, which halts translation (Olsen et al., 1999; Segger erential expression of these inhibitory receptors in normal son et al., 2002). siRNA molecules also are processed by tissues suggests that TRAIL may be useful as an anticancer Dicer, but from a long, double-stranded RNA molecule. siR agent that induces apoptosis in cancer cells while sparing NAs are not naturally found in animal cells, but they can normal cells. (Marsters et al., 1999). direct the sequence-specific cleavage of an mRNA target 0162 There have been many advances in the therapy of through a RNA-induced silencing complex (RISC) (Denliet cancer following the introduction of cytotoxic chemothera al., 2003). peutic drugs. However, one of the consequences of chemo (0169. A. Array Preparation therapy is the development/acquisition of drug-resistant phe 0170 Certain embodiments of the present invention con notypes and the development of multiple drug resistance. The cerns the preparation and use of mRNA or nucleic acid arrays, development of drug resistance remains a major obstacle in miRNA or nucleic acid arrays, and/or miRNA or nucleic acid the treatment of such tumors and therefore, there is an obvi probe arrays, which are macroarrays or microarrays of ous need for alternative approaches Such as gene therapy. nucleic acid molecules (probes) that are fully or nearly 0163 Another form of therapy for use in conjunction with complementary (over the length of the prove) or identical chemotherapy, radiation therapy or biological therapy (over the length of the prove) to a plurality of nucleic acid, includes hyperthermia, which is a procedure in which a mRNA or miRNA molecules, precursor miRNA molecules, patient's tissue is exposed to high temperatures (up to 106° or nucleic acids derived from the various genes and gene F.). External or internal heating devices may be involved in pathways modulated by miR-20 miRNAs and that are posi the application of local, regional, or whole-body hyperther tioned on a Support or Support material in a spatially separated mia. Local hyperthermia involves the application of heat to a organization. Macroarrays are typically sheets of nitrocellu Small area, such as a tumor. Heat may be generated externally lose or nylon upon which probes have been spotted. Microar with high-frequency waves targeting a tumor from a device rays position the nucleic acid probes more densely such that US 2009/0163434 A1 Jun. 25, 2009 74 up to 10,000 nucleic acid molecules can be fit into a region large number of different probes can occupy a relatively small typically 1 to 4 square centimeters. Microarrays can be fab area providing a high density array having a probe density of ricated by spotting nucleic acid molecules, e.g., genes, oligo generally greater than about 60, 100, 600, 1000, 5,000, nucleotides, etc., onto Substrates or fabricating oligonucle 10,000, 40,000, 100,000, or 400,000 different oligonucle otide sequences in situ on a Substrate. Spotted or fabricated otide probes per cm. nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 non-identical nucleic acid molecules 0.175. The surface area of the array can be about or less per square centimeter or higher, e.g. up to about 100 or even than about 1, 1.6, 2,3,4,5,6,7,8,9, or 10 cm. 1000 per square centimeter. Microarrays typically use coated 0176 Moreover, a person of ordinary skill in the art could glass as the Solid Support, in contrast to the nitrocellulose readily analyze data generated using an array. Such protocols based material offilter arrays. By having an ordered array of are disclosed above, and include information found in WO marker RNA and/or miRNA-complementing nucleic acid 9743450; WO 03023058: WO 03022421; WO 03029485; samples, the position of each sample can be tracked and WO 03067217; WO 03066906; WO 03076928: WO linked to the original sample. 03093810; WO 03100448A1, all of which are specifically 0171 A variety of different array devices in which a plu incorporated by reference. rality of distinct nucleic acid probes are stably associated with (0177 B. Sample Preparation the surface of a solid support are known to those of skill in the art. Useful Substrates for arrays include nylon, glass, metal, (0178. It is contemplated that the RNA and/or miRNA of a plastic, latex, and silicon. Such arrays may vary in a number wide variety of samples can be analyzed using the arrays, of different ways, including average probe length, sequence index of probes, or array technology of the invention. While or types of probes, nature of bond between the probe and the endogenous miRNA is contemplated for use with composi array Surface, e.g. covalent or non-covalent, and the like. The tions and methods of the invention, recombinant miRNA labeling and screening methods of the present invention and including nucleic acids that are complementary oridentical to the arrays are not limited in its utility with respect to any endogenous miRNA or precursor miRNA can also be parameter except that the probes detect miRNA, or genes or handled and analyzed as described herein. Samples may be nucleic acid representative of genes; consequently, methods biological samples, in which case, they can be from biopsy, and compositions may be used with a variety of different fine needle aspirates, exfoliates, blood, tissue, organs, semen, saliva, tears, other bodily fluid, hair follicles, skin, or any types of nucleic acid arrays. sample containing or constituting biological cells, particu 0172 Representative methods and apparatus for preparing larly cancer or hyperproliferative cells. In certain embodi a microarray have been described, for example, in U.S. Pat. ments, samples may be, but are not limited to, biopsy, or cells Nos. 5,143,854:5,202,231:5,242,974:5,288,644; 5,324,633; purified or enriched to some extent from a biopsy or other 5,384,261; 5,405,783; 5,412,087; 5,424, 186; 5,429,807; bodily fluids or tissues. Alternatively, the sample may not be 5,432,049; 5,436,327: 5,445,934; 5,468,613; 5,470,710; a biological sample, but be a chemical mixture. Such as a 5,472,672; 5,492,806; 5,525,464; 5,503,980; 5,510,270; cell-free reaction mixture (which may contain one or more 5,525,464; 5,527,681: 5,529,756; 5,532,128: 5,545,531; biological enzymes). 5,547,839; 5,554,501; 5,556,752; 5,561,071; 5,571,639; 5,580,726; 5,580,732; 5,593,839; 5,599,695; 5,599,672; (0179 C. Hybridization 5,610,287: 5,624,711; 5,631,134, 5,639,603; 5,654,413; 0180. After an array or a set of probes is prepared and/or 5,658,734; 5,661,028; 5.665,547; 5,667,972; 5,695,940; the nucleic acid in the sample or probe is labeled, the popu 5,700,637; 5,744,305; 5,800,992; 5,807,522; 5,830,645; lation of target nucleic acids is contacted with the array or 5,837, 196; 5,871,928; 5,847,219; 5,876,932; 5,919,626; probes under hybridization conditions, where such condi 6,004,755; 6,087,102; 6,368,799; 6,383,749; 6,617, 112: tions can be adjusted, as desired, to provide for an optimum 6,638,717; 6,720,138, as well as WO 93/17126; WO level of specificity in view of the particular assay being per 95/11995; WO95/21265; WO95/21944; WO95/35505; WO formed. Suitable hybridization conditions are well known to 96/31622; WO 97/10365; WO 97/27317; WO99/35505; WO those of skill in the art and reviewed in Sambrook et al. (2001) 09923256; WO 09936760; WO0138580; WO 0168255; WO and WO95/2 1944. Of particular interest in many embodi 03020898; WO 03040410; WO 03053586: WO 03087297; ments is the use of stringent conditions during hybridization. WO 03091426; WO03100012: WO 04020085; WO Stringent conditions are known to those of skill in the art. 04027093: EP 373 203; EP 785 280; EP 799 897 and UK 8 0181. It is specifically contemplated that a single array or 803 000; the disclosures of which are all herein incorporated set of probes may be contacted with multiple samples. The by reference. samples may be labeled with different labels to distinguish 0173 It is contemplated that the arrays can be high density the samples. For example, a single array can be contacted arrays, such that they contain 2, 20, 25, 50, 80, 100 or more with a tumor tissue sample labeled with Cy3, and normal different probes. It is contemplated that they may contain tissue sample labeled with Cy5. Differences between the 1000, 16,000, 65,000, 250,000 or 1,000,000 or more different samples for particular miRNAS corresponding to probes on probes. The probes can be directed to mRNA and/or miRNA the array can be readily ascertained and quantified. targets in one or more different organisms or cell types. The 0182. The small surface area of the array permits uniform oligonucleotide probes range from 5 to 50, 5 to 45, 10 to 40, hybridization conditions, such as temperature regulation and 9 to 34, or 15 to 40 nucleotides in length in some embodi salt content. Moreover, because of the small area occupied by ments. In certain embodiments, the oligonucleotide probes the high density arrays, hybridization may be carried out in are 5, 10, 15, 20 to 20, 25, 30, 35, 40 nucleotides in length extremely small fluid volumes (e.g., about 250 ul or less, including all integers and ranges there between. including volumes of about or less than about 5, 10, 25, 50. 0.174. The location and sequence of each different probe 60, 70, 80,90, 100 ul, or any range derivable therein). In small sequence in the array are generally known. Moreover, the Volumes, hybridization may proceed very rapidly. US 2009/0163434 A1 Jun. 25, 2009

0183 D. Differential Expression Analyses during treatment to determine if there are miRNA or genes 0184 Arrays of the invention can be used to detect differ whose expression correlates with the outcome of the patient’s ences between two samples. Specifically contemplated appli treatment. Identification of differential miRNAs or genes can cations include identifying and/or quantifying differences lead to a diagnostic assay for evaluation of tumor and/or blood between miRNA or gene expression from a sample that is samples to determine what drug regimen the patient should be normal and from a sample that is not normal, between a provided. In addition, it can be used to identify or select disease or condition and a cell not exhibiting Such a disease or patients suitable for a particular clinical trial. If an expression condition, or between two differently treated samples. Also, profile is determined to be correlated with drug efficacy or miRNA or gene expression may be compared between a drug toxicity that profile is relevant to whether that patient is sample believed to be susceptible to a particular disease or an appropriate patient for receiving a drug, for receiving a condition and one believed to be not susceptible or resistant to combination of drugs, or for a particular dosage of the drug. that disease or condition. A sample that is not normal is one 0189 In addition to the above prognostic assay, samples exhibiting phenotypic or genotypic trait(s) of a disease or from patients with a variety of diseases can be evaluated to condition, or one believed to be not normal with respect to determine if different diseases can be identified based on that disease or condition. It may be compared to a cell that is miRNA and/or related gene expression levels. A diagnostic normal with respect to that disease or condition. Phenotypic assay can be created based on the profiles that doctors can use traits include symptoms of, or Susceptibility to, a disease or to identify individuals with a disease or who are at risk to condition of which a component is or may or may not be develop a disease. Alternatively, treatments can be designed genetic, or caused by a hyperproliferative or neoplastic cellor based on miRNA profiling. Examples of such methods and cells. compositions are described in the U.S. Provisional Patent 0185. An array comprises a solid support with nucleic acid Application entitled “Methods and Compositions Involving probes attached to the Support. Arrays typically comprise a miRNA and miRNA Inhibitor Molecules' filed on May 23, plurality of different nucleic acid probes that are coupled to a 2005, which is hereby incorporated by reference in its surface of a substrate in different, known locations. These entirety. arrays, also described as “microarrays' or colloquially (0190. E. Other Assays “chips' have been generally described in the art, for example, 0191 In addition to the use of arrays and microarrays, it is U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677, 195, contemplated that a number of different assays could be 6,040,193, 5,424,186 and Fodoret al., (1991), each of which employed to analyze miRNAs or related genes, their activi is incorporated by reference in its entirety for all purposes. ties, and their effects. Such assays include, but are not limited Techniques for the synthesis of these arrays using mechanical to, nucleic acid amplification, polymerase chain reaction, synthesis methods are described in, e.g., U.S. Pat. No. 5,384, quantitative PCR, RT-PCR, in situ hybridization, Northern 261, incorporated herein by reference in its entirety for all hybridization, hybridization protection assay (HPA) (Gen purposes. Although a planar array Surface is used in certain Probe), branched DNA (bDNA) assay (Chiron), rolling circle aspects, the array may be fabricated on a Surface of virtually amplification (RCA), single molecule hybridization detec any shape or even a multiplicity of Surfaces. Arrays may be tion (US Genomics), Invader assay (ThirdWave Technolo nucleic acids on beads, gels, polymeric Surfaces, fibers such gies), and/or Bridge Litigation Assay (Genaco). as fiber optics, glass or any other appropriate Substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 IV. Nucleic Acids and 5,800,992, which are hereby incorporated in their entirety 0.192 The present invention concerns nucleic acids, modi for all purposes. Arrays may be packaged in Such a manner as fied or mimetic nucleic acids, miRNAS, mRNAS, genes, and to allow for diagnostics or other manipulation of an all inclu representative fragments thereof that can be labeled, used in sive device, see for example, U.S. Pat. Nos. 5.856,174 and array analysis, or employed in diagnostic, therapeutic, or 5,922,591 incorporated in their entirety by reference for all prognostic applications, particularly those related to patho purposes. See also U.S. patent application Ser. No. 09/545, logical conditions such as cancer. The molecules may have 207, filed Apr. 7, 2000 for additional information concerning been endogenously produced by a cell, or been synthesized or arrays, their manufacture, and their characteristics, which is produced chemically or recombinantly. They may be isolated incorporated by reference in its entirety for all purposes. and/or purified. Each of the miRNAs described herein and 0186 Particularly, arrays can be used to evaluate samples include the corresponding SEQID NO and accession num with respect to pathological condition such as cancer and bers for these miRNA sequences. The name of a miRNA is related conditions. It is specifically contemplated that the often abbreviated and referred to without a “hsa- prefix and invention can be used to evaluate differences between stages will be understood as such, depending on the context. Unless or Sub-classifications of disease, such as between benign, otherwise indicated, miRNAs referred to in the application cancerous, and metastatic tissues or tumors. are human sequences identified as miR-X or let-X, where X is 0187 Phenotypic traits to be assessed include character a number and/or letter. istics Such as longevity, morbidity, expected Survival, Suscep 0193 In certain aspects, a miRNA probe designated by a tibility or receptivity to particular drugs or therapeutic treat Suffix '5P or “3P can be used. “5P indicates that the ments (drug efficacy), and risk of drug toxicity. Samples that mature miRNA derives from the 5' end of the precursor and a differ in these phenotypic traits may also be evaluated using corresponding “3P indicates that it derives from the 3' end of the compositions and methods described. the precursor, as described on the world wide web at sanger. 0188 In certain embodiments, miRNA and/or expression ac.uk. Moreover, in some embodiments, a miRNA probe is profiles may be generated to evaluate and correlate those used that does not correspond to a known human miRNA. It profiles with pharmacokinetics or therapies. For example, is contemplated that these non-human miRNA probes may be these profiles may be created and evaluated for patient tumor used in embodiments of the invention or that there may exist and blood samples prior to the patient's being treated or a human miRNA that is homologous to the non-human US 2009/0163434 A1 Jun. 25, 2009 76 miRNA. In other embodiments, any mammaliancell, biologi name may or may not be lowercase; for example, hsa-mir cal sample, or preparation thereof may be employed. 130b can also be referred to as miR-130B. The term “miRNA 0194 In some embodiments of the invention, methods and probe' refers to a nucleic acid probe that can identify a par compositions involving miRNA may concern miRNA, mark ticular miRNA or structurally related miRNAs. ers (mRNAs), and/or other nucleic acids. Nucleic acids may 0197) It is understood that some nucleic acids are derived be, beat least, or beat most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, from genomic sequences or a gene. In this respect, the term 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 'gene' is used for simplicity to refer to the genomic sequence 31, 32,33, 34,35,36, 37,38, 39, 40, 41, 42,43, 44, 45, 46,47, encoding the precursor nucleic acid or miRNA for a given 48,49, 50, 51, 52,53,54, 55,56, 57,58, 59, 60, 61, 62,63, 64, miRNA or gene. However, embodiments of the invention may 65, 66,67,68, 69,70, 71,72, 73,74, 75,76, 77,78, 79,80, 81, involve genomic sequences of a miRNA that are involved in 82, 83, 84,85, 86, 87, 88, 89,90,91, 92,93,94, 95, 96, 97,98, its expression, Such as a promoter or other regulatory 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, Sequences. 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 0198 The term “recombinant may be used and this gen 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, erally refers to a molecule that has been manipulated in vitro 360, 370, 380,390, 400, 410, 420, 430, 440, 450, 460, 470, or that is a replicated or expressed product of such a molecule. 480,490,500,510,520, 530, 540, 550,560,570,580, 590, (0199 The term “nucleic acid is well known in the art. A 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, “nucleic acid as used herein will generally refer to a mol 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, ecule (one or more strands) of DNA, RNA or a derivative or 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, analog thereof, comprising a nucleobase. A nucleobase 960, 970, 980, 990, or 1000 nucleotides, or any range deriv includes, for example, a naturally occurring purine or pyri able therein, in length. Such lengths cover the lengths of midine base found in DNA (e.g., an adenine “A” a guanine processed miRNA, miRNA probes, precursor miRNA, “G” a thymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, miRNA containing vectors, mRNA, mRNA probes, control an uracil “U” or a C). The term “nucleic acid encompasses nucleic acids, and other probes and primers. the terms "oligonucleotide' and “polynucleotide.” each as a 0.195. In many embodiments, miRNA are 19-24 nucle subgenus of the term “nucleic acid.” otides in length, while miRNA probes are 19-35 nucleotides 0200. The term “miRNA generally refers to a single in length, depending on the length of the processed miRNA Stranded molecule, but in specific embodiments, molecules and any flanking regions added. miRNA precursors are gen implemented in the invention will also encompass a region or erally between 62 and 110 nucleotides in humans. an additional strand that is partially (between 10 and 50% 0196. Nucleic acids of the invention may have regions of complementary across length of strand), Substantially identity or complementarity to another nucleic acid. It is (greater than 50% but less than 100% complementary across contemplated that the region of complementarity or identity length of strand) or fully complementary to another region of can be at least 5 contiguous residues, though it is specifically the same single-stranded molecule or to another nucleic acid. contemplated that the region is, is at least, or is at most 6, 7, 8, Thus, miRNA may encompass a molecule that comprises one 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more complementary or self-complementary Strand(s) or 26, 27, 28, 29, 30, 31, 32,33, 34, 35,36, 37,38, 39, 40, 41, 42, “complement(s) of a particular sequence. For example, pre 43,44, 45,46, 47, 48,49, 50, 51, 52,53,54, 55,56, 57,58, 59, cursor miRNA may have a self-complementary region, which 60, 61, 62,63, 64, 65, 66, 67,68, 69,70, 71,72, 73,74, 75,76, is up to 100% complementary. miRNA probes or nucleic 77,78, 79,80, 81, 82, 83, 84,85, 86, 87, 88,89,90,91, 92,93, acids of the invention can include, can be or can be at least 60, 94, 95, 96, 97,98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 65, 70, 75, 80, 85,90, 95, 96, 97, 98, 99 or 100% comple 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, mentary to their target. 300, 310,320, 330, 340, 350, 360, 370, 380,390, 400, 410, 0201 It is understood that a “synthetic nucleic acid of the 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510,520, invention means that the nucleic acid does not have all or part 530, 540, 550,560,570, 580,590, 600, 610, 620, 630, 640, of a chemical structure or sequence of a naturally occurring 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, nucleic acid. Consequently, it will be understood that the term 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, “synthetic miRNA refers to a “synthetic nucleic acid' that 890,900,910,920,930,940,950,960,970,980,990, or 1000 functions in a cell or under physiological conditions as a contiguous nucleotides. It is further understood that the naturally occurring miRNA. length of complementarity within a precursor miRNA or 0202 While embodiments of the invention may involve other nucleic acid or between a miRNA probe and a miRNA synthetic miRNAS or synthetic nucleic acids, in some or a miRNA gene are Such lengths. Moreover, the comple embodiments of the invention, the nucleic acid molecule(s) mentarity may be expressed as a percentage, meaning that the need not be “synthetic.” In certain embodiments, a non-syn complementarity between a probe and its target is 90% or thetic nucleic acid or miRNA employed in methods and com greater over the length of the probe. In some embodiments, positions of the invention may have the entire sequence and complementarity is or is at least 90%. 95% or 100%. In structure of a naturally occurring mRNA or miRNA precursor particular, Such lengths may be applied to any nucleic acid or the mature mRNA or miRNA. For example, non-synthetic comprising a nucleic acid sequence identified in any of SEQ miRNAs used in methods and compositions of the invention ID NO: 1 through SEQID NO:269, accession number, or any may not have one or more modified nucleotides or nucleotide other sequence disclosed herein. Typically, the commonly analogs. In these embodiments, the non-synthetic miRNA used name of the miRNA is given (with its identifying source may or may not be recombinantly produced. In particular in the prefix, for example, "hsa' for human sequences) and embodiments, the nucleic acid in methods and/or composi the processed miRNA sequence. Unless otherwise indicated, tions of the invention is specifically a synthetic miRNA and a miRNA without a prefix will be understood to refer to a not a non-synthetic miRNA (that is, not an miRNA that quali human miRNA. Moreover, a lowercase letter in a miRNA fies as “synthetic'); though in other embodiments, the inven US 2009/0163434 A1 Jun. 25, 2009 77 tion specifically involves a non-synthetic miRNA and not a tion of formamide, tetramethylammonium chloride or other synthetic miRNA. Any embodiments discussed with respect solvent(s) in a hybridization mixture. to the use of synthetic miRNAs can be applied with respect to 0207. It is also understood that these ranges, compositions non-synthetic miRNAs, and vice versa. and conditions for hybridization are mentioned by way of 0203. It will be understood that the term “naturally occur non-limiting examples only, and that the desired stringency ring refers to something found in an organism without any for a particular hybridization reaction is often determined intervention by a person; it could refer to a naturally-occur empirically by comparison to one or more positive or negative ring wildtype or mutant molecule. In some embodiments a controls. Depending on the application envisioned it is pre synthetic miRNA molecule does not have the sequence of a ferred to employ varying conditions of hybridization to naturally occurring miRNA molecule. In other embodiments, achieve varying degrees of selectivity of a nucleic acid a synthetic miRNA molecule may have the sequence of a towards a target sequence. In a non-limiting example, identi naturally occurring miRNA molecule, but the chemical struc fication of a related target nucleic acid that does not hybridize ture of the molecule, particularly in the part unrelated specifi to a nucleic acid under Stringent conditions may be achieved cally to the precise sequence (non-sequence chemical struc by hybridization at low temperature and/or high ionic ture) differs from chemical structure of the naturally strength. Such conditions are termed “low stringency” or occurring miRNA molecule with that sequence. In some “low stringency conditions.” and non-limiting examples of cases, the synthetic miRNA has both a sequence and non low stringency include hybridization performed at about 0.15 sequence chemical structure that are not found in a naturally M to about 0.9 MNaCl at a temperature range of about 20°C. occurring miRNA. Moreover, the sequence of the synthetic to about 50° C. Of course, it is within the skill of one in the art molecules will identify which miRNA is effectively being to further modify the low or high Stringency conditions to provided or inhibited; the endogenous miRNA will be Suite a particular application. referred to as the “corresponding miRNA. Corresponding 0208 A. Nucleobase. Nucleoside, Nucleotide, and Modi miRNA sequences that can be used in the context of the fied Nucleotides invention include, but are not limited to, all or a portion of 0209. As used herein a “nucleobase' refers to a heterocy those sequences in SEQID NOs: 1-269, as well as any other clic base. Such as for example a naturally occurring nucleo miRNA sequence, miRNA precursor sequence, or any base (i.e., an A. T. G., C or U) found in at least one naturally sequence complementary thereof. In some embodiments, the occurring nucleic acid (i.e., DNA and RNA), and naturally or sequence is or is derived from or contains all or part of a non-naturally occurring derivative(s) and analogs of Such a sequence identified herein to target a particular miRNA (or nucleobase. A nucleobase generally can form one or more set of miRNAs) that can be used with that sequence. Any 1, 2, hydrogen bonds (“anneal or “hybridize') with at least one 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, naturally occurring nucleobase in a manner that may substi 40, 50, 60, 70, 80,90, 100, 110, 120, 130 140, 150, 160, 170, tute for naturally occurring nucleobase pairing (e.g., the 180, 190, 200,210, 220, 230, 240,250, 260 or any number or hydrogen bonding between A and T G and C, and A and U). range of sequences there between may be selected to the 0210) “Purine” and/or “pyrimidine' nucleobase(s) exclusion of all non-selected sequences. encompass naturally occurring purine and/or pyrimidine 0204 As used herein, “hybridization”, “hybridizes” or nucleobases and also derivative(s) and analog(s) thereof, “capable of hybridizing is understood to mean the forming including but not limited to, those a purine or pyrimidine of a double or triple stranded molecule or a molecule with Substituted by one or more of an alkyl, caboxyalkyl, amino, partial double or triple stranded nature. The term “anneal as hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol used herein is synonymous with “hybridize.” The term or alkylthiol moiety. Preferred alkyl (e.g., alkyl, caboxyalkyl, “hybridization”, “hybridize(s) or “capable of hybridizing” etc.) moieties comprise of from about 1, about 2, about 3, encompasses the terms 'stringent condition(s) or “high about 4, about 5, to about 6 carbon atoms. Other non-limiting stringency” and the terms “low stringency” or “low strin examples of a purine or pyrimidine include a deazapurine, a gency condition(s). 2,6-diaminopurine, a 5-fluorouracil, a Xanthine, a hypoxan 0205 As used herein “stringent condition(s) or “high thine, a 8-bromoguanine, a 8-chloroguanine, a bromothym stringency” are those conditions that allow hybridization ine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylgua between or within one or more nucleic acid strand(s) contain nine, a 8-thioguanine, an azaguanine, a 2-aminopurine, a ing complementary sequence(s), but preclude hybridization 5-ethylcytosine, a 5-methylcyosine, a 5-bromouracil, a of random sequences. Stringent conditions tolerate little, if 5-ethyluracil, a 5-iodouracil, a 5-chlorouracil, a 5-propylu any, mismatch between a nucleic acid and a target Strand. racil, a thiouracil, a 2-methyladenine, a methylthioadenine, a Such conditions are well known to those of ordinary skill in N,N-diemethyladenine, an azaadenines, a 8-bromoadenine, a the art, and are preferred for applications requiring high selec 8-hydroxyadenine, a 6-hydroxyaminopurine, a 6-thiopurine, tivity. Non-limiting applications include isolating a nucleic a 4-(6-aminohexyl/cytosine), and the like. Other examples acid, such as a gene or a nucleic acid segment thereof, or are well known to those of skill in the art. detecting at least one specific mRNA transcript or a nucleic 0211. As used herein, a “nucleoside” refers to an indi acid segment thereof, and the like. vidual chemical unit comprising a nucleobase covalently 0206 Stringent conditions may comprise low salt and/or attached to a nucleobase linker moiety. A non-limiting high temperature conditions, such as provided by about 0.02 example of a “nucleobase linker moiety' is a Sugar compris M to about 0.5 M NaCl at temperatures of about 42° C. to ing 5-carbon atoms (i.e., a "5-carbon Sugar), including but about 70° C. It is understood that the temperature and ionic not limited to a deoxyribose, a ribose, an arabinose, or a strength of a desired stringency are determined in part by the derivative or an analog of a 5-carbon Sugar. Non-limiting length of the particular nucleic acid(s), the length and nucleo examples of a derivative or an analog of a 5-carbon Sugar base content of the target sequence(s), the charge composi include a 2'-fluoro-2'-deoxyribose or a carbocyclic Sugar tion of the nucleic acid(s), and to the presence or concentra where a carbon is substituted for an oxygenatom in the Sugar US 2009/0163434 A1 Jun. 25, 2009

ring. Different types of covalent attachment(s) of a nucleo linking group. The functional moiety and any linker cannot base to a nucleobase linker moiety are known in the art substantially impair the ability of the nucleotide to be added (Kornberg and Baker, 1992). to the miRNA or to be labeled. Representative linking groups 0212. As used herein, a “nucleotide' refers to a nucleoside include carbon containing linking groups, typically ranging further comprising a “backbone moiety'. A backbone moiety from about 2 to 18, usually from about 2 to 8 carbon atoms, generally covalently attaches a nucleotide to another mol where the carbon containing linking groups may or may not ecule comprising a nucleotide, or to another nucleotide to include one or more heteroatoms, e.g. S. O. Netc., and may or form a nucleic acid. The “backbone moiety” in naturally may not include one or more sites of unsaturation. Of particu occurring nucleotides typically comprises a phosphorus moi lar interest in many embodiments are alkyl linking groups, ety, which is covalently attached to a 5-carbon Sugar. The typically lower alkyl linking groups of 1 to 16, usually 1 to 4 attachment of the backbone moiety typically occurs at either carbon atoms, where the linking groups may include one or the 3'- or 5'-position of the 5-carbon sugar. However, other more sites of unsaturation. The functionalized nucleotides (or types of attachments are known in the art, particularly when a primers) used in the above methods of functionalized target nucleotide comprises derivatives or analogs of a naturally generation may be fabricated using known protocols or pur occurring 5-carbon Sugar or phosphorus moiety. chased from commercial vendors, e.g., Sigma, Roche, 0213. A nucleic acid may comprise, or be composed Ambion, Biosearch Technologies and NEN. Functional entirely of a derivative or analog of a nucleobase, a nucleo groups may be prepared according to ways known to those of base linker moiety and/or backbone moiety that may be skill in the art, including the representative information found present in a naturally occurring nucleic acid. RNA with in U.S. Pat. Nos. 4,404,289; 4,405,711; 4,337,063 and 5,268, nucleic acid analogs may also be labeled according to meth 486, and U.K. Patent 1,529.202, which are all incorporated by ods of the invention. As used herein a "derivative' refers to a reference. chemically modified or altered form of a naturally occurring 0217 Amine-modified nucleotides are used in several molecule, while the terms “mimic' or “analog refer to a embodiments of the invention. The amine-modified nucle molecule that may or may not structurally resemble a natu otide is a nucleotide that has a reactive amine group for rally occurring molecule or moiety, but possesses similar attachment of the label. It is contemplated that any ribonucle functions. As used herein, a "moiety’ generally refers to a otide (G, A, U, or C) or deoxyribonucleotide (G, A, T, or C) Smaller chemical or molecular component of a larger chemi can be modified for labeling. Examples include, but are not cal or molecular structure. Nucleobase, nucleoside and nucle limited to, the following modified ribo- and deoxyribo-nucle otide analogs or derivatives are well known in the art, and otides: 5-(3-aminoallyl)-UTP; 8-(4-amino)butyl-amino have been described (see for example, Scheit, 1980, incorpo ATP and 8-(6-amino)butyl-amino-ATP: N6-(4-amino)bu rated herein by reference). tyl-ATP N6-(6-amino)butyl-ATP, N4-2.2-oxy-bis 0214. Additional non-limiting examples of nucleosides, (ethylamine)-CTP; N6-(6-Amino)hexyl-ATP; 8-(6- nucleotides or nucleic acids include those in: U.S. Pat. Nos. Amino)hexyl-amino-ATP: 5-propargylamino-CTP, 5,681,947, 5,652,099 and 5,763,167, 5,614,617, 5,670.663, 5-propargylamino-UTP; 5-(3-aminoallyl)-dUTP; 8-(4- 5,872,232, 5,859,221, 5,446,137, 5,886,165, 5,714,606, amino)butyl-amino-dATP and 8-(6-amino)butyl-amino 5,672,697, 5,466,786, 5,792,847, 5,223,618, 5,470,967, dATP; N6-(4-amino)butyl-dATP, N6-(6-amino)butyl-dATP, 5,378,825, 5,777,092, 5,623,070, 5,610,289, 5,602,240, N4-2.2-oxy-bis-(ethylamine)-dCTP; N6-(6-Amino)hexyl 5,858,988, 5,214,136, 5,700,922, 5,708,154, 5,728,525, dATP; 8-(6-Amino)hexyl-amino-dATP; 5-propargy 5,637,683, 6,251,666, 5,480,980, and 5,728,525, each of lamino-dCTP, and 5-propargylamino-dUTP. Such nucle which is incorporated herein by reference in its entirety. otides can be prepared according to methods known to those 0215 Labeling methods and kits of the invention specifi of skill in the art. Moreover, a person of ordinary skill in the cally contemplate the use of nucleotides that are both modi art could prepare other nucleotide entities with the same fied for attachment of a label and can be incorporated into a amine-modification, such as a 5-(3-aminoallyl)-CTP, GTP. miRNA molecule. Such nucleotides include those that can be ATP, dCTP, dGTP, dTTP, or dUTP in place of a 5-(3-aminoal labeled with a dye, including a fluorescent dye, or with a lyl)-UTP. molecule such as biotin. Labeled nucleotides are readily 0218 B. Preparation of Nucleic Acids available; they can be acquired commercially or they can be 0219. A nucleic acid may be made by any technique synthesized by reactions known to those of skill in the art. known to one of ordinary skill in the art, Such as for example, 0216 Modified nucleotides for use in the invention are not chemical synthesis, enzymatic production, or biological pro naturally occurring nucleotides, but instead, refer to prepared duction. It is specifically contemplated that miRNA probes of nucleotides that have a reactive moiety on them. Specific the invention are chemically synthesized. reactive functionalities of interest include: amino, sulfhydryl, 0220. In some embodiments of the invention, miRNAs are Sulfoxyl, aminosulfhydryl, azido, epoxide, isothiocyanate, recovered or isolated from a biological sample. The miRNA isocyanate, anhydride, monochlorotriazine, dichlorotriazine, may be recombinant or it may be natural or endogenous to the mono- or dihalogen Substituted pyridine, mono- or disubsti cell (produced from the cell's genome). It is contemplated tuted diazine, maleimide, epoxide, aziridine, Sulfonylhalide, that a biological sample may be treated in a way so as to acid halide, alkyl halide, aryl halide, alkylsulfonate, N-hy enhance the recovery of small RNA molecules such as droxysuccinimide ester, imido ester, hydrazine, azidonitro miRNA. U.S. patent application Ser. No. 10/667,126 phenyl, azide, 3-(2-pyridyl dithio)-propionamide, glyoxal, describes Such methods and it is specifically incorporated by aldehyde, iodoacetyl, cyanomethyl ester, p-nitrophenyl ester, reference herein. Generally, methods involve lysing cells o-nitrophenyl ester, hydroxypyridine ester, carbonyl imida with a solution having guanidinium and a detergent. Zole, and the other Such chemical groups. In some embodi 0221 Alternatively, nucleic acid synthesis is performed ments, the reactive functionality may be bonded directly to a according to standard methods. See, for example, Itakura and nucleotide, or it may be bonded to the nucleotide through a Riggs (1980) and U.S. Pat. Nos. 4,704,362, 5,221,619, and US 2009/0163434 A1 Jun. 25, 2009 79

5,583,013, each of which is incorporated herein by reference. Materials for implementing tube electrophoresis can be Non-limiting examples of a synthetic nucleic acid (e.g., a readily prepared by a person of skill in the art or purchased, synthetic oligonucleotide), include a nucleic acid made by in such as from C.B.S. Scientific Co., Inc. or Scie-Plas. vitro chemically synthesis using phosphotriester, phosphite, 0228 Methods may involve the use of organic solvents or phosphoramidite chemistry and Solid phase techniques and/or alcohol to isolate nucleic acids, particularly miRNA such as described in EP 266.032, incorporated herein by used in methods and compositions of the invention. Some reference, or via deoxynucleoside H-phosphonate intermedi embodiments are described in U.S. patent application Ser. ates as described by Froehler et al., 1986 and U.S. Pat. No. No. 10/667,126, which is hereby incorporated by reference. 5,705,629, each incorporated herein by reference. Various Generally, this disclosure provides methods for efficiently different mechanisms of oligonucleotide synthesis have been isolating Small RNA molecules from cells comprising: add disclosed in for example, U.S. Pat. Nos. 4,659.774, 4,816, ing an alcohol solution to a cell lysate and applying the 571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, alcohol/lysate mixture to a solid support before eluting the 5,574,146, 5,602.244, each of which is incorporated herein RNA molecules from the solid support. In some embodi by reference. ments, the amount of alcohol added to a cell lysate achieves 0222. A non-limiting example of an enzymatically pro an alcohol concentration of about 55% to 60%. While differ duced nucleic acid include one produced by enzymes in ent alcohols can be employed, ethanol works well. A solid amplification reactions such as PCRTM (see for example, U.S. Support may be any structure, and it includes beads, filters, Pat. Nos. 4,683.202 and 4,682,195, each incorporated herein and columns, which may include a mineral or polymer Sup by reference), or the synthesis of an oligonucleotide port with electronegative groups. A glass fiber filter or column described in U.S. Pat. No. 5,645,897, incorporated herein by has worked particularly well for such isolation procedures. reference. See also Sambrook et al., 2001, incorporated 0229. In specific embodiments, miRNA isolation pro herein by reference). cesses include: a) lysing cells in the sample with a lysing 0223 Oligonucleotide synthesis is well known to those of Solution comprising guanidinium, wherein a lysate with a skill in the art. Various different mechanisms of oligonucle concentration of at least about 1 M guanidinium is produced; otide synthesis have been disclosed in for example, U.S. Pat. b) extracting miRNA molecules from the lysate with an Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, extraction Solution comprising phenol; c) adding to the lysate 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is an alcohol solution for forming a lysatefalcohol mixture, incorporated herein by reference. wherein the concentration of alcohol in the mixture is 0224 Recombinant methods for producing nucleic acids between about 35% to about 70%; d) applying the lysate/ in a cell are well known to those of skill in the art. These alcohol mixture to a solid support; e) eluting the miRNA include the use of vectors (viral and non-viral), plasmids, molecules from the Solid Support with an ionic Solution; and, cosmids, and other vehicles for delivering a nucleic acid to a f) capturing the miRNA molecules. Typically the sample is cell, which may be the target cell (e.g., a cancer cell) or simply dried and resuspended in a liquid and Volume appropriate for a host cell (to produce large quantities of the desired RNA Subsequent manipulation. molecule). Alternatively, such vehicles can be used in the context of a cell free system so long as the reagents for V. Labels and Labeling Techniques generating the RNA molecule are present. Such methods 0230. In some embodiments, the present invention con include those described in Sambrook, 2003, Sambrook, 2001 cerns miRNA that are labeled. It is contemplated that miRNA and Sambrook, 1989, which are hereby incorporated by ref may first be isolated and/or purified prior to labeling. This CCC. may achieve a reaction that more efficiently labels the 0225 C. Isolation of Nucleic Acids miRNA, as opposed to other RNA in a sample in which the 0226 Nucleic acids may be isolated using techniques well miRNA is not isolated or purified prior to labeling. In many known to those of skill in the art, though in particular embodi embodiments of the invention, the label is non-radioactive. ments, methods for isolating Small nucleic acid molecules, Generally, nucleic acids may be labeled by adding labeled and/or isolating RNA molecules can be employed. Chroma nucleotides (one-step process) or adding nucleotides and tography is a process often used to separate or isolate nucleic labeling the added nucleotides (two-step process). acids from protein or from other nucleic acids. Such methods 0231 A. Labeling Techniques can involve electrophoresis with a gel matrix, filter columns, 0232. In some embodiments, nucleic acids are labeled by alcohol precipitation, and/or other chromatography. If catalytically adding to the nucleic acid an already labeled miRNA from cells is to be used or evaluated, methods gen nucleotide or nucleotides. One or more labeled nucleotides erally involve lysing the cells with a chaotropic (e.g., guani can be added to miRNA molecules. See U.S. Pat. No. 6,723, dinium isothiocyanate) and/or detergent (e.g., N-lauroylsar 509, which is hereby incorporated by reference. cosine) prior to implementing processes for isolating 0233. In other embodiments, an unlabeled nucleotide or particular populations of RNA. nucleotides is catalytically added to a miRNA, and the unla 0227. In particular methods for separating miRNA from beled nucleotide is modified with a chemical moiety that other nucleic acids, a gel matrix is prepared using polyacry enables it to be subsequently labeled. In embodiments of the lamide, though agarose can also be used. The gels may be invention, the chemical moiety is a reactive amine Such that graded by concentration or they may be uniform. Plates or the nucleotide is an amine-modified nucleotide. Examples of tubing can be used to hold the gel matrix for electrophoresis. amine-modified nucleotides are well known to those of skill Usually one-dimensional electrophoresis is employed for the in the art, many being commercially available Such as from separation of nucleic acids. Plates are used to prepare a slab Ambion, Sigma, Jena BioScience, and TriLink. gel, while the tubing (glass or rubber, typically) can be used to 0234. In contrast to labeling of cDNA during its synthesis, prepare a tube gel. The phrase “tube electrophoresis” refers to the issue for labeling miRNA is how to label the already the use of a tube or tubing, instead of plates, to form the gel. existing molecule. The present invention concerns the use of US 2009/0163434 A1 Jun. 25, 2009

an enzyme capable of using a di- or tri-phosphate ribonucle Blue-7-dUTP, Alexa Fluor 488-5-dUTP. Fluorescein-12 otide or deoxyribonucleotide as a substrate for its addition to dUTP, Oregon Green 488-5-dUTP BODIPY FL-14-dUTP, a miRNA. Moreover, in specific embodiments, it involves Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, using a modified di- or tri-phosphate ribonucleotide, which is BODIPY TMR-14-dUTP. Tetramethylrhodamine-6-dUTP. added to the 3' end of a miRNA. Enzymes capable of adding Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Such nucleotides include, but are not limited to, poly(A)poly Red-12-dUTP, Texas Red-5-dUTP BODIPY TR-14-dUTP, merase, terminal transferase, and polynucleotide phosphory Alexa Fluor 594-5-dUTP BODIPY 630/650-14-dUTP, lase. In specific embodiments of the invention, a ligase is BODIPY 650/665-14-dUTP; Alexa Fluor 488-7-OBEA contemplated as not being the enzyme used to add the label, dCTP, Alexa Fluor 546-16-OBEA-dCTP, Alexa Fluor 594-7- and instead, a non-ligase enzyme is employed. Terminal OBEA-dCTP, Alexa Fluor 647-12-OBEA-dCTP. transferase catalyzes the addition of nucleotides to the 3' 0241. It is contemplated that nucleic acids may be labeled terminus of a nucleic acid. Polynucleotide phosphorylase can with two different labels. Furthermore, fluorescence reso polymerize nucleotide diphosphates without the need for a nance energy transfer (FRET) may be employed in methods primer. of the invention (e.g., Klostermeier et al., 2002; Emptage, 0235 B. Labels 2001; Didenko, 2001, each incorporated by reference). 0236 Labels on miRNA or miRNA probes may be colo 0242 Alternatively, the label may not be detectable perse, rimetric (includes visible and UV spectrum, including fluo but indirectly detectable or allowing for the isolation or sepa rescent), luminescent, enzymatic, or positron emitting (in ration of the targeted nucleic acid. For example, the label cluding radioactive). The label may be detected directly or could be biotin, digoxigenin, polyvalent cations, chelator indirectly. Radioactive labels include ‘’I, ‘P. P. and S. groups and the other ligands, include ligands for an antibody. Examples of enzymatic labels include alkaline phosphatase, 0243 C. Visualization Techniques luciferase, horseradish peroxidase, and B-galactosidase. 0244. A number of techniques for visualizing or detecting Labels can also be proteins with luminescent properties, e.g., labeled nucleic acids are readily available. Such techniques green fluorescent protein and phicoerythrin. include, microscopy, arrays, Fluorometry, Light cyclers or 0237. The colorimetric and fluorescent labels contem other real time PCR machines, FACS analysis, scintillation plated for use as conjugates include, but are not limited to, counters, Phosphoimagers, Geiger counters, MRI. CAT, anti Alexa Fluor dyes, BODIPY dyes, such as BODIPY FL: Cas body-based detection methods (Westerns, immunofluores cade Blue; Cascade Yellow; coumarin and its derivatives, cence, immunohistochemistry), histochemical techniques, such as 7-amino-4-methylcoumarin, aminocoumarin and HPLC (Griffey et al., 1997), spectroscopy, capillary gel elec hydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins trophoresis (Cummins et al., 1996), spectroscopy; mass spec and erythrosins; fluorescein and its derivatives. Such as fluo troscopy; radiological techniques; and mass balance tech rescein isothiocyanate; macrocyclic chelates of lanthanide niques. ions, such as Quantum DyeTM: Marina Blue: Oregon Green; 0245. When two or more differentially colored labels are rhodamine dyes, such as rhodamine red, tetramethyl employed, fluorescent resonance energy transfer (FRET) rhodamine and rhodamine 6G, Texas Red; fluorescent energy techniques may be employed to characterize association of transfer dyes. Such as thiazole orange-ethidium heterodimer; one or more nucleic acid. Furthermore, a person of ordinary and, TOTAB. skill in the art is well aware of ways of visualizing, identify 0238 Specific examples of dyes include, but are not lim ing, and characterizing labeled nucleic acids, and accord ited to, those identified above and the following: Alexa Fluor ingly, such protocols may be used as part of the invention. 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Examples of tools that may be used also include fluorescent Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor 532, Alexa microscopy, a BioAnalyzer, a plate reader, Storm (Molecular Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor Dynamics), Array Scanner, FACS (fluorescent activated cell 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Sorter), or any instrument that has the ability to excite and Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, detect a fluorescent molecule. Alexa Fluor 750; amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, VI. Kits BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, 0246. Any of the compositions described herein may be BODIPY 630/650, BODIPY 650/655, BODIPY FL, comprised in a kit. In a non-limiting example, reagents for BODIPY R6G, BODIPY TMR, and, BODIPY-TR: Cy3, isolating miRNA, labeling miRNA, and/or evaluating a Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, miRNA population using an array, nucleic acid amplification, Oregon Green 488, Oregon Green 500, Oregon Green 514, and/or hybridization can be included in a kit, as well reagents Pacific Blue, REG, Rhodamine Green, Rhodamine Red, for preparation of samples from blood samples. The kit may Renographin, ROX, SYPRO, TAMRA, 2',4',5'7"-Tetrabro further include reagents for creating or synthesizing miRNA mosulfonefluorescein, and TET. probes. The kits will thus comprise, in suitable container 0239 Specific examples of fluorescently labeled ribo means, an enzyme for labeling the miRNA by incorporating nucleotides are available from Molecular Probes, and these labeled nucleotide or unlabeled nucleotides that are subse include, Alexa Fluor 488-5-UTP, Fluorescein-12-UTP, quently labeled. In certain aspects, the kit can include ampli BODIPY FL-14-UTP, BODIPYTMR-14-UTP. Tetramethyl fication reagents. In other aspects, the kit may include various rhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5- Supports, such as glass, nylon, polymeric beads, and the like, UTP, and BODIPY TR-14-UTP. Other fluorescent ribonucle and/or reagents for coupling any probes and/or target nucleic otides are available from Amersham Biosciences, such as acids. It may also include one or more buffers. Such as reac Cy3-UTP and Cy5-UTP. tion buffer, labeling buffer, washing buffer, or a hybridization 0240 Examples of fluorescently labeled deoxyribonucle buffer, compounds for preparing the miRNA probes, and otides include Dinitrophenyl (DNP)-11-dUTP. Cascade components for isolating miRNA. Other kits of the invention US 2009/0163434 A1 Jun. 25, 2009 may include components for making a nucleic acid array 0252) However, the components of the kit may be pro comprising miRNA, and thus, may include, for example, a vided as dried powder(s). When reagents and/or components Solid Support. are provided as a dry powder, the powder can be reconstituted 0247 Kits for implementing methods of the invention by the addition of a suitable solvent. It is envisioned that the Solvent may also be provided in another container means. In described herein are specifically contemplated. In some Some embodiments, labeling dyes are provided as a dried embodiments, there are kits for preparing miRNA for multi power. It is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, labeling and kits for preparing miRNA probes and/or miRNA 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, arrays. In these embodiments, kit comprise, in Suitable con 300, 400, 500, 600, 700, 800, 900, 1000 ug or at least or at tainer means, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more of the most those amounts of dried dye are provided in kits of the following: (1) poly(A) polymerase; (2) unmodified nucle invention. The dye may then be resuspended in any Suitable otides (G. A. T. C. and/or U); (3) a modified nucleotide solvent, such as DMSO. (labeled or unlabeled); (4) poly(A) polymerase buffer; and, 0253 Such kits may also include components that facili (5) at least one microfilter; (6) label that can be attached to a tate isolation of the labeled miRNA. It may also include nucleotide; (7) at least one miRNA probe; (8) reaction buffer; components that preserve or maintain the miRNA or that (9) a miRNA array or components for making such an array; protect against its degradation. Such components may be (10) acetic acid; (11) alcohol; (12) solutions for preparing, RNAse-free or protect against RNAses. Such kits generally isolating, enriching, and purifying miRNAS or miRNA will comprise, in Suitable means, distinct containers for each probes or arrays. Other reagents include those generally used individual reagent or solution. for manipulating RNA, such as formamide, loading dye, ribo 0254. A kit will also include instructions for employing nuclease inhibitors, and DNase. the kit components as well the use of any other reagent not 0248. In specific embodiments, kits of the invention included in the kit. Instructions may include variations that include an array containing miRNA probes, as described in can be implemented. the application. An array may have probes corresponding to 0255 Kits of the invention may also include one or more all known miRNAS of an organism or a particular tissue or of the following: Control RNA; nuclease-free water; RNase organ in particular conditions, or to a Subset of Such probes. free containers, such as 1.5 ml tubes; RNase-free elution The subset of probes on arrays of the invention may be or tubes; PEG or dextran; ethanol; acetic acid; sodium acetate; include those identified as relevant to a particular diagnostic, ammonium acetate; guanidinium; detergent; nucleic acid size therapeutic, or prognostic application. For example, the array marker; RNase-free tube tips; and RNase or DNase inhibi may contain one or more probes that is indicative or Sugges tOrS. tive of (1) a disease or condition (acute myeloid leukemia), 0256. It is contemplated that such reagents are embodi (2) Susceptibility or resistance to a particular drug or treat ments of kits of the invention. Such kits, however, are not ment; (3) Susceptibility to toxicity from a drug or Substance; limited to the particular items identified above and may (4) the stage of development or severity of a disease or con include any reagent used for the manipulation or character dition (prognosis); and (5) genetic predisposition to a disease ization of miRNA. or condition. 0249 For any kit embodiment, including an array, there VII. EXAMPLES can be nucleic acid molecules that contain or can be used to 0257 The following examples are included to demon amplify a sequence that is a variant of identical to or comple strate preferred embodiments of the invention. It should be mentary to all or part of any of SEQID NOS: 1-267. In certain appreciated by those of skill in the art that the techniques embodiments, a kit or array of the invention can contain one disclosed in the examples which follow represent techniques or more probes for the miRNAs identified by SEQID NOS: discovered by the inventor to function well in the practice of 1-267. Any nucleic acid discussed above may be imple the invention, and thus can be considered to constitute pre mented as part of a kit. ferred modes for its practice. However, those of skill in the art 0250. The components of the kits may be packaged either should, in light of the present disclosure, appreciate that many in aqueous media or in lyophilized form. The container means changes can be made in the specific embodiments which are of the kits will generally include at least one vial, test tube, disclosed and still obtain a like or similar result without flask, bottle, Syringe or other container means, into which a departing from the spirit and scope of the invention. component may be placed, and preferably, Suitably aliquoted. Where there is more than one component in the kit (labeling Example 1 reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container Gene Expression Analysis Following Transfection into which the additional components may be separately with HSA-MiR-20a placed. However, various combinations of components may 0258 miRNAs are believed to regulate gene expression by be comprised in a vial. The kits of the present invention also binding to target mRNA transcripts and (1) initiating tran will typically include a means for containing the nucleic Script degradation or (2) altering protein translation from the acids, and any other reagent containers in close confinement transcript. Translational regulation leading to an up or down for commercial sale. Such containers may include injection or change in protein expression may lead to changes in activity blow molded plastic containers into which the desired vials and expression of downstream gene products and genes that are retained. are in turn regulated by those proteins. These numerous regu 0251 When the components of the kit are provided in one latory effects may be revealed as changes in the global mRNA and/or more liquid solutions, the liquid Solution is an aqueous expression profile. Microarray gene expression analyses were Solution, with a sterile aqueous solution being particularly performed to identify genes that are mis-regulated by hsa preferred. miR-20a expression. US 2009/0163434 A1 Jun. 25, 2009

0259 Synthetic Pre-miR-20a (Ambion) was reverse trans cancers. Manipulation of the expression levels of genes in the fected into quadruplicate samples of A549 cells for each of cellular pathways shown in Table 2 represents a potentially three time points. Cells were transfected using siPORT useful therapy for cancer and other diseases in which NeoFX (Ambion) according to the manufacturer's recom increased or reduced expression of hsa-miR-20a has a role in mendations using the following parameters: 200,000 cells per the disease. well in a 6 well plate, 5.0 ul of NeoFX, 30 nM final concen tration of miRNA in 2.5 ml. Cells were harvested at 4h, 24h, Example 3 and 72 h post transfection. Total RNA was extracted using RNAqueous-4PCR (Ambion) according to the manufactur Predicted Gene Targets of Hsa-MiR-20a er's recommended protocol. 0264 Gene targets for binding of and regulation by hsa 0260 mRNA array analyses were performed by Asuragen miR-20a were predicted using the proprietary algorithm Services (Austin,Tex.), according to the company's standard miRNA TargetTM (Asuragen) and are shown in Table 3 supra. operating procedures. Using the MessageAmpTMII-96 aRNA 0265. The predicted gene targets that exhibited altered Amplification Kit (Ambion, cat #1819) 2 ug of total RNA mRNA expression levels in human cancer cells, following were used for target preparation and labeling with biotin. transfection with pre-miRhsa-miR-20a, are shown in Table 4 cRNA yields were quantified using an Agilent Bioanalyzer Supra. 2100 capillary electrophoresis protocol. Labeled target was 0266 The predicted gene targets of hsa-miR-20a whose hybridized to Affymetrix mRNA arrays (Human HG-U133A mRNA expression levels are affected by hsa-miR-20a repre 2.0 arrays) using the manufacturer's recommendations and sent particularly useful candidates for cancer therapy and the following parameters. Hybridizations were carried out at therapy of other diseases through manipulation of their 45° C. for 16 hr in an Affymetrix Model 640 hybridization expression levels. oven. Arrays were washed and stained on an Affymetrix FS450 Fluidics station, running the wash script Midi Example 4 euk2v3 450. The arrays were scanned on a Affymetrix GeneChip Scanner 3000. Summaries of the image signal Cancer Related Gene Expression Altered by HSA data, group mean values, p-values with significance flags, log MiR-20a ratios and gene annotations for every gene on the array were 0267 Cell proliferation and survival pathways are com generated using the Affymetrix Statistical Algorithm MAS monly altered in tumors (Hanahan and Weinberg, 2000). The 5.0 (GCOS v 1.3). Data were reported in a file (cabinet) con inventors have shown that hsa-miR-20a directly or indirectly taining the Affymetrix data and result files and in files (cel) regulates the transcripts of proteins that are critical in the containing the primary image and processed cell intensities of regulation of these pathways. Many of these targets have the arrays. Data were normalized for the effect observed by inherent oncogenic or tumor Suppressor activity. Hsa-miR the average of two negative control microRNA sequences and 20a targets that are associated with various cancer types are then were averaged together for presentation. A list of genes shown in Table 5. whose expression levels varied by at least 0.7 log from the 0268 Hsa-miR-20a targets of particular interest are genes average negative control was assembled. Results of the and their products that function in the regulation of intracel microarray gene expression analysis are shown in Table 1 lular signal transduction. When deregulated, many of these Supra. proteins contribute to the malignant phenotype in vitro and in 0261 Manipulation of the expression levels of the genes vivo. Hsa-miR-20a affects intracellular signaling at various listed in Table 1 represents a potentially useful therapy for layers and controls the expression of secretory growth factors, cancer and other diseases in which increased or reduced transmembrane growth factor receptors, and cytoplasmic sig expression of hsa-miR-20a has a role in the disease. naling molecules. Examples of secreted proteins regulated by hsa-miR-20a are Eregulin (EREG), Wnt5a and the inflamma Example 2 tory chemokine IL-8. Eregulin (EREG) belongs to the epi Cellular pathways affected by HSA-MiR-20a dermal growth factor (EGF) family and binds to EGF recep tors such as ErbB (Shelly et al., 1998). Eregulin expression is 0262 The mis-regulation of gene expression by hsa-miR rare in adult tissues but is elevated in various cancer types 20a (Table 1) affects many cellular pathways that represent (Toyoda et al., 1997). Eregulin may also play a direct role in potential therapeutic targets for the control of cancer and tumorigenesis, as it contributes to tumor formation of colon other diseases and disorders. The inventors determined the cancer cells (Baba et al., 2000). Since transfection of hsa identity and nature of the cellular genetic pathways affected miR-20a decreases levels of EREG transcripts, hsa-miR-20a by the regulatory cascade induced by hsa-miR-20a expres might intervene with the oncogenic activity of Eregulin. Wnt Sion. Cellular pathway analyses were performed using Inge family members are cysteine-rich proteins that function as nuity Pathways Analysis (Ingenuity(R) Systems, Redwood growth factors. Wnt5a plays a role in patterning decisions in City, Calif.). The most significantly affected pathways fol the embryonic nervous system during development and is lowing over-expression of hsa-miR-20a in A549 cells are linked to the progression of melanoma and the invasion of shown in Table 2 supra. ductal breast carcinomas (Jonsson et al., 2002; Weeraratna et 0263. These data demonstrate that hsa-miR-20a directly al., 2002). Transmembrane receptors targeted by hsa-miR or indirectly affects the expression of numerous cellular 20a include platelet-derived growth factor receptor-like growth-, cellular proliferation-, cell signaling-, and cell (PDGFR-L, also known as PDGF-receptor beta-like tumor development-related genes and thus primarily affects func suppressor, PRLTS), transforming growth factor beta (TGF tional pathways related to, cellular growth, cellular develop B) receptor 2 (TGFBR2), tumor necrosis factor-related apo ment, and cell proliferation. Those cellular processes all have ptosis inducer ligand (TRAIL) receptor 2 (TRAIL-R2; also integral roles in the development and progression of various known as tumor necrosis factor receptor Superfamily member US 2009/0163434 A1 Jun. 25, 2009

B10; TNFSFB10), retinoic acid receptor responder 1 the cell cycle. Cyclin D1 is required for the transition from G1 (RARRES1), ephrin B2 receptor (EphB2) and fibroblast into S phase and is overexpressed in numerous cancer types growth factor receptors (FGFR) 3 and 4. FGFR-3 and (Donnellan and Chetty, 1998). Hsa-miR-20a negatively regu FGFR-4 are commonly overexpressed in multiple cancer lates cyclin D1 expression and therefore might interfere with types and appear to have angiogenic activity (Chandler et al., abnormal cell growth that depends on high levels of cyclin 1999). In contrast, PDGFR-L, TRAIL-R2, RARRES1 and D1. In contrast, cyclin G1 has growth inhibitory activity and TGFBR-2 are putative tumor suppressors. PDGFR-L shows is upregulated by hsa-miR-20a (Zhao et al., 2003). Skp2 is a loss of function in a broad variety of cancers either by loss of component of the multi-subunit E3 ubiquitin ligase complex heterozygosity (LOH) or missense and frame-shift mutation that ear-marks proteins for proteasomal degradation. A well (Fujiwara et al., 1995; Komiya et al., 1997). TRAIL-R2 inter characterized target is the CDK inhibitor p27 which offers an acts with TRAIL and stimulates pro-apoptotic pathways in explanation for the cell cycle promoting activity of Skp2 various cell types (Fesik, 2005). The corresponding gene is (Carrano et al., 1999). Skp2 is inherently oncogenic and located at a chromosomal region (8p22-23) that is a frequent shows elevated levels in various cancer types (Gstaiger et al., site of LOH in numerous human neoplasias (Adams et al., 2001; Kamata et al., 2005; Saigusa et al., 2005; Einama et al., 2005). Therefore, loss of TRAIL-R2 may contribute to the 2006). malignant phenotype of these cancers. 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- Continued <213> ORGANISM: Rattus norvegicus <4 OO SEQUENCE: 23 ulaaagugcuul aulagugcagg ulag 23

<210 SEQ ID NO 24 <211 LENGTH: 21 &212> TYPE : RNA <213> ORGANISM: Rattus norvegicus

<4 OO SEQUENCE: 24 actugcaululac gag cacuulac a 21

<210 SEQ ID NO 25 <211 LENGTH: 21 &212> TYPE : RNA <213> ORGANISM: Rattus norvegicus <4 OO SEQUENCE: 25 caaagugcluc aulagugcagg u. 21

<210 SEQ ID NO 26 <211 LENGTH: 21 &212> TYPE : RNA <213> ORGANISM: Rattus norvegicus <4 OO SEQUENCE: 26 actugcagugu gag cacuucu g 21

<210 SEQ ID NO 27 <211 LENGTH: 22 &212> TYPE : RNA <213> ORGANISM: Saguinus labiatus

<4 OO SEQUENCE: 27 ulaaagugcuul aulagugcagg ula 22

<210 SEQ ID NO 28 <211 LENGTH: 22 &212> TYPE : RNA <213> ORGANISM: Sus scrofa

<4 OO SEQUENCE: 28 ulaaagugcuul aulagugcagg ula 22

<210 SEQ ID NO 29 <211 LENGTH: 23 &212> TYPE : RNA <213> ORGANISM: Tetraodon nigroviridis

<4 OO SEQUENCE: 29 ulaaagugcuul aulagugcagg ulag 23

<210 SEQ ID NO 3 O <211 LENGTH: 23 &212> TYPE : RNA <213> ORGANISM: Xenopus laevis <4 OO SEQUENCE: 30 caaagugcluc aulagugcagg ulag 23