Clin Genet 2008: 74: 296–306 # 2008 The Authors Printed in Singapore. All rights reserved Journal compilation # 2008 Blackwell Munksgaard CLINICAL GENETICS doi: 10.1111/j.1399-0004.2008.01076.x Review in development and disease

Erson AE, Petty EM. MicroRNAs in development and disease. AE Ersona and EM Pettyb,c # Clin Genet 2008: 74: 296–306. Blackwell Munksgaard, 2008 aDepartment of Biological Sciences, Middle East Technical University (METU), Since the discovery of microRNAs (miRNAs) in Caenorhabditis elegans, Ankara, Turkey, and bDepartment of mounting evidence illustrates the important regulatory roles for miRNAs Internal Medicine and cDepartment of in various developmental, differentiation, cell proliferation, and Human Genetics, University of Michigan, apoptosis pathways of diverse organisms. We are just beginning to MI, USA elucidate novel aspects of RNA mediated regulation and to understand how heavily various molecular pathways rely on miRNAs for their normal function. miRNAs are small non--coding transcripts that regulate gene expression post-transcriptionally by targeting Key words: cancer – development – messenger RNAs (mRNAs). While individual miRNAs have been microRNA – viral infection specifically linked to critical developmental pathways, the deregulated Corresponding author: Elizabeth M Petty, expression of many miRNAs also has been shown to have functional Department of Internal Medicine, significance for multiple human diseases, such as cancer. We continue to University of Michigan, 5220 MSRB III, discover novel functional roles for miRNAs at a rapid pace. Here, we 1150 West Medical Center Drive, Ann summarize some of the key recent findings on miRNAs, their mode of Arbor, MI 48109-0640, USA. action, and their roles in both normal development and in human Tel.: 1(734) 763-2532; pathology. A better understanding of how miRNAs operate during the fax: 1(734) 647-7979; normal life of a cell as well as in the pathogenesis of disease when e-mail: [email protected] deregulated will provide new avenues for diagnostic, prognostic, and Received 2 May 2008, revised and therapeutic applications. accepted for publication 3 July 2008

When the Project was com- been widely investigated in various eukaryotic or- pleted, the number of in our genome was ganisms (1–3). By 2002, the miRNA registry (ver- estimated to be around 20–25 000, far fewer than sion 1) had 218 miRNA entries for primates, the initially predicted 100 000. All known or pre- rodents, birds, fish, worms, flies, plants and vi- dicted protein-coding genes are thought to be ex- ruses. This number dramatically increased over pressed only from a small percentage (i.e. 1.5–2%) 6 years to 6396 as of April 2008 (version 11) (4– of the whole genome. These figures are poor in- 6). This noteworthy discovery rate during a 6-year dicators of the actual genetic complexity in hu- time period along with the increasing evidence for mans. In fact, limiting the definition of Ôgene’ to the regulatory roles of many miRNAs in various describe only protein-coding units is not compre- cellular processes, such as development and differ- hensive enough either, to define genetic complex- entiation, are clear indicators of the considerable ity. As of today, the majority of the human complexity of genetic information processing in genome is hypothesized to code for many non- cells. Current estimations predict that about protein coding structural and regulatory RNAs, 30% of all the human genes are regulated by miR- including microRNAs (miRNAs), that play criti- NAs (7) and that a single miRNA can potentially cal and vital roles in generating genomic complex- target around 200 different transcripts that may ity and diversity within Homo sapiens and, also, function in different pathways in the cell (8). between species. The nomenclature for miRNAs has yet to be miRNAs are small non-protein-coding tran- fully standardized in the literature, but, in general, scripts of 16–29 nucleotide-long RNAs that regu- miRNA genes are designated by the italicized pre- late gene expression post-transcriptionally by fix Ômir’. The unitalicized prefix ÔmiR’ is used to targeting messenger RNAs (mRNAs). Since the indicate the mature form of the miRNA. The pre- initial discovery in Caenorhabditis elegans in fixes Ômir’ or ÔmiR’ are followed in most cases by 1993, miRNA-dependent gene regulation has a dash and then a number, such as miR-127. A set

296 MiRNAs in development and disease of guidelines for miRNA annotation is suggested are also likely to be transcribed by RNA polymer- by Ambros et al. (9). ase III (11). Given the large number of recent papers in the The general miRNA biogenesis pathway (Fig. 1) scientific literature describing the functional roles involves a primary miRNA (pri-miRNA) tran- of miRNAs in biological processes such as devel- script (several hundred base pairs to several kilo- opment, differentiation, cell proliferation, and base pairs) that is subsequently cleaved by the apoptosis, it is clear that miRNAs are critical reg- nuclear RNase III enzyme, , and its partner ulatory factors important for normal health and DGCR8 (DiGeorge syndrome critical region gene are also highly relevant to disease processes. Here, 8) forming the 60–70 nucleotide-long precursor we present a brief outline of miRNA biogenesis miRNA (pre-miRNA) with a 3# overhang of two and action mechanisms with an emphasis on key nucleotides (12, 13). The pre-miRNA is then trans- roles of miRNAs in normal development and ported to the cytoplasm through the nuclear export human pathology. protein, exportin-5, together with Ran-guanosine triphosphate (14). Cytoplasmic RNase III, , and its RNA-binding partner TRBP, human immunodeficiency virus (HIV)-1 transactivating miRNA biogenesis responsive element (TAR) RNA-binding protein, Similar to mRNAs, miRNAs can be transcribed in cleave the pre-miRNA into a 20–25 nucleotide a time- and tissue-specific manner. Many miR- mature miRNA duplex. This double-stranded NAs are transcribed by RNA polymerase II and RNA then assembles into a multiprotein complex, have a poly(A) tail and a 5# cap (10). However, RNA-induced silencing complex (RISC), which is exceptions do exist. For instance, Borchert et al. composed of Dicer, TRBP, and Argonaute 2 demonstrated polymerase III dependent tran- (Ago2) (13, 15–17). Caudy and Hannon (18) also scription of a human 19 miR cluster showed presence of Ago2, Drosophila fragile X- (C19MC). miR-515-1, miR-517a, miR-517c and related protein, vasa intronic gene and the micro- miR-519a-1, as well as other miRNAs with coccal nuclease family member Tudor-SN (Dro- upstream tRNA, repeat or transposable elements, sophila CG7008) in the Drosophila RISC structure.

Fig. 1. Overview of the microRNA (miRNA) biogenesis pathway. Up to several kb long pri-miRNA is usually transcribed by RNA polymerase II, has a 5#cap and 3#poly(A) tail. Pri-miRNAs fold into stem loop structures that are cleaved by nuclear RNase III Drosha and its RNA-binding partner DGCR8 (DiGeorge syndrome critical region gene 8) into 60–70 nucleotide long pre-miRNA, leaving a two-nucleotide 3# overhang which is then transported to the nucleus through exportin 5, Ran- GTP. Cytoplasmic RNase III, Dicer, and its RNA-binding partner TRBP cleave the pre-miRNA into a 20–25 nucleotide mature miRNA duplex. The double-stranded RNA then assembles into the RNA-induced silencing complex (RISC). One of the strands is degraded, whereas the strand harboring the mature miRNA and the RISC complex is directed to the target mRNA. At this point, different routes are known to exist for gene silencing; translational repression or mRNA degradation (see text for details).

297 Erson and Petty RISC brings the target mRNA and the mature miR-1224 and miR-1225) in mammals and 16 pri- miRNA strand (also known as the Ôguide’ strand) mate-specific mirtrons have been identified (29). together while causing removal of the Ôpassenger’ miRNAs can also be found in exons of non-coding strand (19). The selection of the guide or the pas- RNAs [e.g. miR-155 is encoded within an exon of senger strands is currently thought to be based on the non-coding RNA known as B-cell integration the thermodynamic characteristics of the strands. cluster] (30, 31). It has been generally accepted that the less stable Relatively less is known about the transcrip- strand becomes the guide strand and is the target tional regulation of intergenic or intronic (if dif- mRNA-binding miRNA while the more stable pas- ferent than the host gene) miRNAs. Some recent senger strand is removed from the complex for deg- data showed that c-myc induced transcription of radation. On the contrary, however, Ro et al. (20) a miRNA cluster, miR-17-92 (32), (33) and p53 provides evidence that both strands of the miRNA induced expression of miR-34 genes (reviewed in duplex (sister strands) can indeed be functional and 34). Not surprisingly, expression of miRNAs is possibly co-accumulate in a tissue-dependent man- also shown to be affected by epigenetic mecha- ner to target different mRNA populations. How- nisms. Saito et al. (35) demonstrated increased ever, the mechanism of how such specific expression of miR-127, along with 16 of 313 other regulation takes place is yet to be examined. human miRNAs examined, after DNA methyla- Interestingly, there seems to be additional fine tion and histone deactylase inhibitor treatments. tuning mechanisms to manage the biogenesis of After treatment, proto-oncogene BCL6, a poten- miRNAs. For example, Lin 28, a developmentally tial target of miR-127, was translationally down- regulated RNA-binding protein, has been recently regulated, reportedly because of the increased shown to selectively block let-7 miRNA process- expression of the miR-127 (also see 36 for a review ing (potentially by binding to the terminal loop of epigenetically regulated miRNAs). region of let-7 precursors) in embryonic cells, thus, acting as a negative regulator (21, 22). On the contrary, transforming growth factor b and Mode of action bone morphogenetic protein-specific SMAD sig- nal transducers have been detected in a complex miRNAs generally bind to their target mRNAs’ 3# with pri-miR-21 and the RNA helicase p68 (also untranslated region (UTR) and most often lead to known as DDX5), a component of the DROSHA, a decrease of the target protein by either degrada- possibly enhancing the processing of pri-miRNA- tion of the target mRNA or translational repres- 21 into pre-miRNA-21, leading to an increase in sion. In some cases, both mechanisms seem to be mature miR-21 levels (23). operational. The extent of base pairing between miRNAs can be expressed as mono- or polycis- the miRNA and mRNA seems to be an important tronic transcripts. They can be found located determinant for the ultimate fate of the target either in intergenic regions (13), potentially with mRNA. Usually perfect, or near perfect, their own transcriptional start sites, CpG islands, complementation results in mRNA decay. For transcription factor binding sites and poly(A) sig- example; miR-196, important during differentia- nals (24) or embedded within the introns of pro- tion, binds to the 3#UTR of the HoxB8 mRNA tein-coding genes (25), where they exhibit with almost perfect complementation and cause a concurrent expression profile with the host gene miRNA-dependent cleavage of the mRNA within (26) [also see Li et al. (27) for a list of human and the miRNA-binding site (37). For this type of en- mouse intronic miRNAs]. However, it is also pos- donucleolytic cleavage, also known as Slicer activ- sible that some intronic miRNAs are in antisense ity, presence of a specific argonaute protein, Ago2 orientation to the host gene or, alternatively, that that has an RNaseH-like domain is also function- the size of intron may be large enough to harbor ally required in the RISC complex (38). However, independent regulatory units for the miRNA gene cleavage does not always take place within the within the intron. In this case, the expression pro- miRNA–mRNA complementarity regions. Bagga file of the intronic miRNA may be independent of et al. (39) reported degradation of lin-41 mRNA, the host gene. As first detected in Drosophila mel- a target of let-7, outside the miRNA complemen- anogaster and C. elegans (28), and later in mam- tary sites. More interestingly, 3#UTR of lin-41 mals, instead of the initial cleavage by Drosha, mRNA was only partially complementary to let-7. some miRNA are spliced out as introns but are Translational silencing or blocking of the target afterwards processed as pre-miRNAs (29). These mRNA, however, usually takes place by the miRNAs originating from an intron are named as imperfect base pairing between one or more Ômirtrons’. At the time of this review, three well- miRNAs (same or different miRNAs) and the conserved and expressed mirtrons (miR-877, 3#UTR region of the target mRNA. Blocking of

298 MiRNAs in development and disease translation is thought to manifest at either the single nucleotide polymorphisms (SNPs) also initiation or elongation stage of translation (40). have interesting implications in target mRNA– Mathonnet et al. (41) elegantly demonstrated in miR binding. Mishra et al. (56) report an SNP, an in vitro system that the 5# cap recognition pro- in the 3#UTR of human dihydrofolate reductase cess is repressed by endogenous let-7 and that sub- (DHFR) gene, which affects DHFR expression by sequent miRNA inhibition of translation interfering with miR-24 binding, resulting in initiation occurs by targeting the cap-binding DHFR overexpression and methotrexate resis- complex eIF4F. miRNA-mediated translation tance (also see 57). repression can also happen through the inhibition Thus, at this time, the full repertoire of mecha- of 60S ribosomal subunit joining to the 40S initi- nisms by which miRNAs regulate gene expression ation complex (42). According to Petersen et al. is likely incomplete and awaits further investiga- (43), imperfect base pairing between short RNAs tion to illustrate the breadth, diversity, and tissue- and the 3#UTR of a target mRNA may also result and temporal-specific functional mechanisms that in ribosomal drop-off at multiple sites within the are critical to cellular function. While our under- open reading frame; therefore, some miRNAs are standing of miRNAs is increasing, new studies proposed to modulate translation through an clearly demonstrate how much more complicated increased rate of termination. miRNA-related pathways in the cell can be. The resulting translationally silenced target An interesting study by Hwang et al. (58) dem- mRNAs are then thought to be sequestered by onstrated that a terminal hexonucleotide sequence the miRNAs to cytoplasmic processing bodies of miR-29b can act as a nuclear localization signal (P-bodies, also known as GW bodies), where for the miRNA. Specific localization of this, or untranslated mRNAs are stored and, ultimately, potentially other miRNAs, to the nucleus may sometimes degraded (44), possibly through activ- imply localization-specific roles/functions of miR- ities of other key that are also localized to NAs in addition to their well-established roles in P-bodies such as Dcp1/Dcp2 (mRNA decapping translational regulation in the cytoplasm. In addi- enzymes) and Xrn1 (exonuclease) (45–47). In fact, tion, the finding that miRNAs may be secreted may argonaute proteins, miRNAs and target mRNAs indicate other as-of-yet unknown roles for some are all found localized to P-bodies (48). Interest- miRNAs, such as exosomal let-7, miR-1, miR-15, ingly, Bhattacharyya et al. (49) demonstrated that miR-16, miR-181 and miR-375 (59). Secreted miR- miR-122-induced inhibition of the cationic amino NAs could potentially even have functions during acid transporter 1 mRNA could be a reversible cell-to-cell communication and interactions; how- process in human hepatoma cells. Further studies ever, such functional roles have not yet been are needed to determine if this reversibility of fate described. Such secreted miRNA-induced interac- is a common or rare outcome of miRNA-induced tions are likely to have relevance during the com- inhibition as well as to fully elucidate the regula- plex events of development and differentiation. tors of miRNA silencing for specific genes. Perhaps one of the most startling findings about Somewhat surprisingly, to date few miRNAs miRNAs came as a surprise when Vasudevan et al. have been shown to be prone to RNA editing. (54) reported that miR-369-3, together with AGO Kawahara et al. (50) demonstrated that RNA ed- (argonaute) and FXR1 (fragile X mental retarda- iting of pri-miR-151 and blockage of its cleavage tion-related protein 1) can bind to the AU-rich by Dicer leads to the accumulation of edited pre- 3#UTR region of tumor necrosis factor-a tran- miR-151 RNAs. Tissue-specific RNA editing script to activate its translation under low serum potentially seems to regulate alternative mRNA:- conditions rather than downregulate as might miRNA interactions as demonstrated by miR-376 have been anticipated. Furthermore, Orom et al. targeting a different set of genes due to RNA edit- (55) provided evidence that mir-10a may posi- ing in different tissues (51). Liang and Landweber tively control protein synthesis via binding to the (52) further speculated that RNA editing of target 5#UTRs of ribosomal mRNAs and enhancing mRNA to alter mRNA:miRNA binding could be their translation. Another type of regulation a regulation mechanism in cells important for where an miRNA can bind to a coding region of gene expression. Another interesting observation an mRNA to control translation, came from that illustrates how much more complicated Duursma et al.’s (60) work that suggested miR- miRNA-mediated regulation of mRNAs could 148 to repress DNA methyltransferase 3b be is based on initial studies that suggested that (Dnmt3b) through binding to a region in its cod- the binding of miRNAs to any position on a target ing sequence. These intriguing findings will help mRNA, other than the 3#UTR, is also mechanis- pave the way for exciting functional studies of tically possible and sufficient for translational miRNAs in disease that may alter the existing repression (53) or activation (54, 55). Moreover, paradigms.

299 Erson and Petty miRNAs in development and diseases wealth of data reported to date clearly indicates that miRNAs have important and critical roles in Dicer knockout (KO) mouse models provided sig- normal development in multiple organ systems nificant evidence for specific roles of miRNAs dur- including the heart and nervous system and can ing mammalian development. The Dicer KO impact human diseases ranging from infectious mouse did not survive beyond 7.5 days past gastru- diseases to cancer. lation (61) suggesting the vital role of miRNAs in early development. Subsequent conditional models with reduced Dicer activity caused various devel- opmental defects in limb growth (62), lung epithe- Cardiovascular pathologies and miRNAs lial morphogenesis (63) and stem cells’ maintenance Both congenital and adult-onset heart diseases (61). While loss of Dicer activity could have had account for significant mortality and morbidity effects other than miRNA biogenesis in these in developed countries. Interestingly, there are models, the initial observations were further con- biological connections between fetal heart devel- firmed by global miRNA expression analysis and opment and adult-onset heart diseases where re- individual characterization of miRNAs solidifying activation of fetal genes can be observed in a role for miRNAs in mammalian development. pathological hypertrophy and heart failure (74). Currently, we are just beginning to understand In addition to transcriptome analysis, miRNA ar- roles of individual miRNAs in invertebrate and rays have also revealed an expression pattern quite vertebrate development and differentiation. For similar to that of fetal cardiac tissue in failing example, stem cell proliferation, division and dif- human myocardium (75), further confirming the ferentiation (e.g. adipocyte, cardiac, neural and need of a precise control of miRNA expression in hematopoietic lineages) are important processes the heart throughout development and in termi- in which investigators have started to pinpoint nally differentiated cardiomyocytes. Various specific miRNA functions (64). Similarly, an studies have revealed pivotal roles of miRNAs in miRNA expression profile has been created for both cardiac structure and function. Among human embryonic stem cell lines by comparing these, miR-1 (also highly expressed in skeletal undifferentiated and differentiated states (64). In muscle), miR-133, miR-208, miR-21 and miR- addition to data from high throughput analyses, 221/222 (also implicated in cell cycle progression miR-520h is linked to differentiation of hemato- and melanoma 76, 77), have validated targets with poietic stem cells into progenitor cells (65). miR- roles in cardiac-related mechanisms such as car- 181, miR-223, and miR-142 have been shown to diac growth, erythropoiesis, angiogenesis, prolif- have roles in differentiation of the hematopoietic eration, etc. (78, 79). Zhao et al. (80) generated lineage (66). miR-150 can direct differentiation of a miR-1-deficient mouse that demonstrated heart megakaryocyte–erythrocyte progenitors into meg- enlargement due to cellular hyperplasia, ventricu- akaryocytes in vitro and in vivo (67). Muscle-specific lar septal defects, abnormalities in electrical con- expression of miR-1, miR-133a and miR-206 sug- duction, and cell cycle control. miR-208 KO mice gested involvement of these miRNAs during myo- provided clues about the involvement of this miR genesis (68–70). Recently, Wong and Tellam (71) in stress-dependent regulation of b-myosin heavy pointed out that miR-26a can promote myogenesis chain, one of the contractile proteins of the heart by repressing a suppressor of skeletal muscle cell (81). Similarly, a heart-specific Dicer KO mouse differentiation, Ezh2, resulting with upregulation demonstrated dilated cardiomyopathy, a sign of of myoD and myogenin mRNA expression. heart failure, with deregulated contractile proteins miRNAs are also rapidly emerging as switches and sarcomere structures (82). From these experi- of key regulatory pathways in the cell, such as ments, it is obvious that deregulation of miRNAs critical alternative splicing mechanisms, which can have profound effects on the cardiovascular may contribute to tissue specificity. For example, system. In addition to the characterized miRNAs muscle-specific miR-133 has been shown to down- so far, other individual miRNAs and their targets regulate the alternative splicing regulator neuro- need to be studied to better understand their roles nal polypyrimidine tract-binding protein during in cardiac development and heart disease. myoblast differentiation to control splicing of cer- tain exon combinations (72). Similarly, Makeyev et al. (73) demonstrated that miR-124 is involved Nervous system development/pathologies in nervous system development by regulating neu- and miRNAs ron-specific alternative splicing. While all the mechanisms important in miRNA regulation of Neuropsychiatric conditions impact the well- development have not yet been elucidated, the being of millions of people worldwide, often

300 MiRNAs in development and disease impacting their productivity and longevity. The impairment with moderate clinical AD, and indi- central nervous system (CNS) is a rich source of viduals with full-blown clinical features of AD. miRNAs, some of which are just specific to brain They reported significantly decreased miR-107 tissue (83 and see 84 for a review). Temporal and levels in patients with even the earliest stages of spatial expression of miRNAs seems to be essen- the disease. They further analyzed the role of this tial for the development and differentiation of miRNA and concluded that miR-107 may be in- CNS in general. Initial evidence on the role of volved in accelerated disease progression through miRNAs in CNS came from Dicer KO models regulation of the beta-site amyloid precursor in zebra fish, which demonstrated abnormal mor- protein-cleaving enzyme 1 (BACE1). Hebert et al. phogenesis during gastrulation, somitogenesis, (95) recently demonstrated that miR-29a, miR- brain and heart development (85). Later studies 29b-1, and miR-9 can regulate BACE1 expression pinpointed individual miRNA expression in dif- in vitro and that these miRNAs were decreased in ferent neural cells of zebra fish, such as miR-92b AD patients resulting with high BACE1 protein. expression in neuronal precursors and stem cells, miRNAs have also been implicated in chronic miR-9 expression in both proliferative cells and psychiatric disorders. With many targets in com- their differentiated progeny, miR-222 expression mon, miR-206 and miR-198 loci were found to be in telencephalon, and miR-218a expression in associated with schizophrenia (96). In addition, motor neurons (86). Additionally, miR-134 has miR-26b, miR-30b, miR-29b, miR-195, miR-92, a demonstrated role in dendritic spine develop- miR-24, miR-30e were shown by microarray and ment (87) and miR140 has proven its importance quantitative reverse transcriptase-polymerase chain in regulating platelet-derived growth factor recep- reaction to be decreased in samples from individ- tor alpha mRNA during cranial neural crest cell uals with schizophrenia (97). These miRNAs and migration in zebra fish (88). their targets and how they may be involved in com- Of these miRNAs, miR-124 is one of the most mon complex neurological and psychiatric disease characterized neuronal miRNAs to date. miR-124 states are yet to be examined. expression has been associated with the transition from proliferation to differentiation as well as constitutive expression in mature neurons (86) Viral pathogenesis and miRNAs and has demonstrated involvement in the brain- specific alternative pre-mRNA splicing, and Viral diseases are an important and growing downregulation of an anti-neuronal factor, small global health problem. Several different lines of C-terminal domain phosphatase 1 (73, 89). Lam- evidence indicate critical involvement of miRNAs inin gamma 1 and integrin beta1, which are highly in viral pathogenesis pathways. Host miRNAs expressed by neural progenitors and repressed may be activated due to viral insertion, viral miR- upon neuronal differentiation, are also key targets NAs may target host mRNAs, viral miRNAs may of miR-124 (90). A recent high throughput anal- target viral mRNAs, or host miRNAs may target ysis further characterized specific miRNA expres- viral mRNAs. Virus integration-induced activa- sion profiles for the distinct areas of the adult tion of host miR-17-92 and miR-106a-363 has mouse CNS such as the spinal cord, cerebellum been shown in virus-induced tumors (98, 99). In and hippocampus (91). All of these findings along addition to the activation of host miRNAs, more with other data in the scientific literature indicate than 100 viral miRNAs that may play roles during that deregulated miRNA expression in the CNS infection have been identified (100). While some may be highly relevant to better understand neu- of these miRNAs target viral mRNAs, some actu- rodegenerative diseases such as Parkinson’s dis- ally target host mRNAs, which likely contribute ease and Alzheimer’s disease (AD). to the development of disease and/or disease vir- A progressive loss of midbrain dopaminergic ulence. Kaposi’s-sarcoma-associated herpes virus neurons (DNs), a Parkinson’s disease-like pheno- (KSHV) is a large DNA virus that has been impli- type, was detected in conditional Dicer KO mice cated in Kaposi’s sarcoma and hyperproliferative in post-mitotic midbrain, which suggested that B-cell diseases. Expression of viral miR-K12-11, miRNAs are essential for the terminal differenti- an orthologue of cellular miR-155, resulted in ation and/or maintenance of multiple neuron the downregulation of key genes with roles in types, including midbrain DNs (92, 93). Wang cell growth regulation. Given the role of miR- et al. (94) analyzed miRNA expression profiles 155 in B-cell transformation, miR-K12-11 may of human brain tissue from non-demented indi- have contributed to the induction of KSHV- viduals with negligible clinical features of AD, positive B-cell tumors in infected patients (101, non-demented individuals with incipient clinical 102). Upregulated miR-155 was also indicated in features of AD, individuals with mild cognitive Burkitt lymphoma (103). Moreover, oncogenic

301 Erson and Petty translocations were detected in a transgenic mice proven more reliable than mRNA profiles to that carries a mutation in the activation-induced define tumor subclasses in some cases (112). One cytidine deaminase (required for immunoglobulin of many such miRNAs identified to be deregu- gene diversification in B lymphocytes) 3#UTR at lated in cancers was miR-21, which was overex- the site of miR-155 binding (104). pressed in a variety of different tumors including Two miRNAs identified in Simian virus 40 breast cancers (for a review see 113). So far, iden- (SV40) suggested targeting of viral mRNAs rather tified targets of miR-21 are PTEN and PDCD4, than host mRNAs (105). SV40 miRNAs accumu- genes that are well-known to be involved in cell late at late times in infection, and target mRNAs survival and transformation processes (114, 115). responsible for the expression of early infection The polycistronic miRNA cluster miR-17-92 (on viral T antigens (106). This provides a means to 13q31.3) is thought to enhance cell proliferation reduce susceptibility of SV40-infected cells to and has been found to be overexpressed in several cytotoxic T lymphocytes that normally target T- tumor types such as lung (116), and, more antigen-harboring cells. Similarly, Epstein–Barr recently, in colorectal cancers (117). virus-encoded miR-BART2 was shown to down- In addition to overexpressed miRNAs that have regulate the viral DNA polymerase BALF5, pos- putative oncogenic roles, specific miRNAs that sibly controlling latent to lytic viral replication as may have tumor suppressor roles have been iden- induction of the lytic viral replication cycle results tified and are found to be downregulated (109). in a reduction of the level of miR-BART2 (107). For example, let-7 is a miRNA that was found Finally, as an example of host miRNAs target- to be downregulated in cancers and it targets the ing viral mRNAs, HIV-1 mRNAs have been widely recognized oncogene, RAS (118). Other shown to be targeted by cellular miRNAs (e.g. investigated targets of let-7 so far are the early miR-28, miR-125b, miR-150, miR-223 and miR- embryonic HMGA2 oncogene (119) and IMP-1 382), which may have roles in HIV-1 latency in (120). IMP-1, an oncofetal gene reexpressed in resting primary CD41 T lymphocytes (108). miR- cancers, stabilizes target RNAs (e.g. c-myc, NAs have not only been implicated in viral path- IGF2 and H19) and protects them from degrada- ogenesis but also during the modulation of tion (121). Yu et al. (122) further showed that let-7 immune response as well as other blood-related regulates differentiation and self-renewal of breast pathologies potentially including autoimmune cells through its targets HMGA2 and RAS in diseases and cancer (82). Therefore, understand- breast cancer cells. ing the role of miRNAs in host, pathogen and Downregulation of miRNAs in tumors led to immune system relationships may open new ave- the hypothesis that epigenetic mechanisms may nues of research and hopefully lead to more effi- turn off transcription of the miRNA genes that cient treatment options. are located in CpG islands. Indeed, hyperme- thylation was shown for miR-9-1, miR-124a3, miR-148, miR-152, and miR-663 in 34–86% of 71 primary human breast cancers (123). miR-34b, miRNAs in cancer miR-137, miR-193a, and miR-203, located A better understanding of the molecular mecha- around CpG islands, were found to be silenced nisms underlying oncogenesis may provide new through aberrant DNA methylation in oral squa- strategies to prevent and manage the morbidity mous cell carcinomas and their expression was and mortality due to cancer. Early clues linking restored by treatment with 5-aza-2#-deoxycytidine miRNAs to cancer came from observations in (5-Aza-CdR) (124). While expressed in normal chronic lymphocytic leukemia (CLL). Chromo- tissues, miR-127 was detected to be downregu- some band 13q14, commonly lost or altered in lated in various cancers (e.g. prostate and bladder) CLL patients, was found to harbor miR-15a and mostly due to increased methylation in tumor cells miR-16a, which turned out to control the antia- (35). Interestingly, when its expression was poptotic B-cell lymphoma protein, BCL-2 in B induced after 5-Aza-CdR and histone deacetylase cells (109, 110). Initial observations that miRNA inhibitor, 4-phenylbutyric acid, expression of genes were located on genomic instability and BCL6, a target of miR-127, was suppressed sug- fragile sites by Calin et al. (111) lead to further gesting a tumor suppressor function for mir-127 analyses that demonstrated deregulated miRNA (35). Another example of an epigenetic regulation expression profiles in various tumors. miRNA ar- for miRNA genes reported in the literature is for rays provided information of deregulated miRNA miR-223. AML1/ETO, an acute myeloid leuke- expression profiles even specific enough to be pre- mia-associated fusion protein due to t(8;21) trans- dictive for tumor classification purposes. In fact, location, recruits chromatin remodeling enzymes differentially expressed miRNA profiles were at an AML1-binding site on the mir-223 gene,

302 MiRNAs in development and disease resulting in the heterochromatic silencing of mir- sity, and disease pathology. Examples of miRNA 223. RNAi against the fusion protein, or demethy- expression profiling experiments have proved to lating agent treatments enhanced miR-223 levels be powerful tools for the identification and classi- and restored cell differentiation (125). fication of tumors as biomarkers. Hence, miRNAs miRNAs can theoretically target more than one are drawing well-deserved attention as potential target mRNA; therefore, a single miRNA can therapeutic targets. Chemically engineered Ôanta- potentially act as both an oncogene, if it targets gomirs’ in mice (132) and locked nucleic acid- an antiproliferative gene, and as a tumor supp- modified oligos in non-human primates (133) seem ressor if it targets a growth-promoting gene. In to be promising for efficient and specific silencing addition, oncogenes and tumor suppressors them- of miRNAs. Therefore, RNA-interference-based selves have been shown to regulate the transcrip- therapeutic efforts are already being developed tion of some miRNA genes. Recently, several using various delivery options (e.g. viral, lipid- groups reported p53-induced miRNA transcrip- based and nano-carriers). Because we are just tion (e.g. miR-34a and miR-34b/c), which may beginning to understand about miRNAs, predic- participate in the p53-regulated apoptosis, cell tion and validation of their target mRNAs, their cycle arrest, and senescence (34, 126). Members modes of action and tissue-specific targeting of of miR-17-92 (miR-17-5p and miR-20a) targeted miRNAs are among some challenges that need E2F1 mRNA while the miR-17-92 cluster itself to be addressed before successful therapeutic ap- was activated by c-myc, which also activates plications (see 134 for a discussion). E2F1 transcription (32). Thus, it seems that c- In time, miRNAs are likely to prove indispens- myc may be controlling E2F1 by both transcrip- able not only as diagnostic, predictive, and prog- tional activation and translational inhibition nostic tools but also are likely to become through miRNAs. The miR-106b-25 cluster was important therapeutic targets for a wide selection also shown to be activated by E2F1 that is later of human pathologies ranging from viral infec- regulated by a negative feedback loop through tions to cancer. miR-106b and miR-93 (127). miRNAs also have been found to play im- perative roles during invasion and metastasis References pathways. miR-10b, regulating migration and 1. Lee R, Ambros V. An extensive class of small RNAs in invasion, was found to be highly expressed in met- Caenorhabditis elegans. Science 2001: 294: 862–864. astatic breast cancer cells compared with non- 2. Ambros V, Lee R, Lavanway A et al. MicroRNAs and other tiny endogenous RNAs in C. elegans. 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