DOCSLIB.ORG

US 2013/0143320 A1 Li Et Al

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
US 2013/0143320 A1 Li Et Al US 2013 O143320A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0143320 A1 Li et al. (43) Pub. Date: Jun. 6, 2013 (54) METHODS AND COMPOSITIONS FOR (60) Provisional application No. 60/392,142, filed on Jun. MANIPULATING THE GUIDED NAVIGATION 27, 2002. OF ENDOTHELLALTUBES DURING ANGIOGENESIS Publication Classification (71) Applicants: Dean Y. Li, Salt Lake City, UT (US); (51) Int. Cl Kye Won Park, Salt Lake City, UT (US) CI2N5/071 (2006.01) (72) Inventors: Dean Y. Li, Salt Lake City, UT (US); C07K 4/705 (2006.01) Kye Won Park, Salt Lake City, UT (US) (52) U.S. Cl CPC ............ CI2N5/0691 (2013.01); C07K 14/705 (73) Assignee: University of Utah Research (2013.01) Foundation, Salt Lake City, UT (US) USPC ........................... 435/375:536/23.5; 530/350 (21) Appl. No.: 13/658,526 (57) ABSTRACT (22) Filed: Oct. 23, 2012 Methods and compositions for manipulating the directed Related U.S. Application Data navigation of physiological tracking tubular structures are .S. App provided. A novel cell-bound receptor, roundabout-4 (Robo (63) Continuation of application No. 12/975,160, filed on 4), is described. The Robo-4 receptor shows sequence and Dec. 21, 2010, now abandoned, which is a continu structural similarity to members of the roundabout family of ation of application No. 10/519,342, filed on Sep. 21, receptors. Also, the Robo-4 receptor binds Slit ligand, a 2005, now abandoned, filed as application No. PCT/ known receptor of the roundabout receptors. Polynucleotides US03/20508 on Jun. 27, 2003. and polypeptides of the Robo-4 receptor are described. Patent Application Publication Jun. 6, 2013 Sheet 1 of 6 US 2013/0143320 A1 Figure 1 embryo YS Ak1 +/- - - - - -l- Kb. 6.5 - 4.9 - 3.6 - east assage Robo4 26 -- 19 - 13 - O.9 - O.6 - 0.2 - Patent Application Publication Jun. 6, 2013 Sheet 2 of 6 US 2013/0143320 A1 Figure 2 E9.O *r Patent Application Publication Jun. 6, 2013 Sheet 3 of 6 US 2013/0143320 A1 Figure 3 M Robo 4 C) () () o a Zf Robo 4 HoH H Robo Ho 2 M. Rig-1 (Robo 3) Ho H. H. H. His Figure 4 fobo. 3 fobo2 fobo Robo 4 hobo 4 mRigi zRobo Robo Robo 1 Robo zRobo:3 2 obo Patent Application Publication Jun. 6, 2013 Sheet 4 of 6 US 2013/0143320 A1 Figure 5 Patent Application Publication Jun. 6, 2013 Sheet 5 of 6 US 2013/0143320 A1 Robo4-HEK Control-HEK 4. 4 g 3 g 3 2 2 2 2 C O 2 1 P 1 O O Control FGF HEKCM Slyc NOne FGF HEKCM Sisye Figure 6A Figure 6B Rob04-HEK Control-HEK 7.5 7.5 3 S; 5.0 50 2 2 C 2.5 C 2.5 f f O None FGF Slit-myc Slit-myc O None FGF Slit-myc Slit-myc CM CM CM CM None N-Robot None N-Robot depletion depletion Figure 60 Figure 6D Robo4-HEK Control-HEK 7.5 7.5 3 8 d 5.0 d 50 c 2.5 c 2.5 f f O Control FGF Slit-myc Slit-myc Slit-myc O None FGF Slit-myc Slit-myc Slit-myc CM CM CM CM CM CM Antic-myc Anti-HA Antic-myc Anti-HA depletion depletion Figure 6E Figure 6F Patent Application Publication Jun. 6, 2013 Sheet 6 of 6 US 2013/0143320 A1 12 8 4 None VEGF HEKCM Slit-myc CMSi-myc CMSlit-myc CMSt-myc CM Anti HA Anti-cmyc N-Robot -- depletion Figure 7 US 2013/0143320 A1 Jun. 6, 2013 METHODS AND COMPOSITIONS FOR 0006. Therefore, there is a need for compositions and MANIPULATING THE GUIDED NAVIGATION methods that are able to manipulate the navigation of physi OF ENDOTHELLALTUBES DURING ological tracking tubular structures, such as endothelial tubes, ANGOGENESIS such that the structures can be directed towards a desired tissue, or prevented from reaching a target tissue. FIELD OF THE INVENTION SUMMARY OF THE INVENTION 0001. The present invention relates to methods and com positions that are useful in manipulating the guidance of 0007. The present invention is directed at a guidance sys physiological tracking tubular structures, such as endothelial tem and methods that function to direct navigation of physi tubes. In preferred embodiments, the invention relates to ological tubular structures, such as endothelial tubes. The methods and compositions that are useful in manipulating the invention includes a novel cell-bound receptor, Roundabout 4 directed navigation of endothelial tubes, such as during (Robo-4), that is expressed in endothelial cells and interacts angiogenesis, during embryonic development and in the with a known ligand to affect directed navigation of endothe neovascularization of tumors and other cell masses and/or lial tubes during vascular development. Together, the Robo-4 tissues. More specifically, the present invention relates to a receptor and the ligand, the slit ligand, inhibit the directed guidance system that can be used to both direct endothelial navigation of endothelial tubes to target cell masses and/or tubes toward a target, such as ischemic tissue, and direct tubes tissues. Thus, the interaction between the Robo-4 receptor away from a target, Such as a solid tumor. and the slit ligand provides a repulsive cue that affects the guidance of tubular structures, such as endothelial tubes. As BACKGROUND OF THE INVENTION described herein, the repulsive cue provided by Robo-4/slit interactions can be used to direct tubular structure both 0002 The vasculature provides a network of vessels that toward and away from a tissue or other cell mass of interest. efficiently delivers nutrients to and removes waste from tis 0008. The present invention is useful for a variety of pur sues of the body. This network extends throughout most of the poses. For example, the polynucleotides of the present inven body, reaching all major tissues, and consists of two distinct tion can be used for gene therapy, such as replacement of types of structures—arteries and veins. The arterial and defective copies of naturally occurring genes or provision of venous systems are parallel networks that function to deliver Supplemental genes. Furthermore, the polypeptides of the blood to a tissue and carry blood and waste away from tissues, present invention can be used in therapeutic procedures. For respectively. These two systems are anatomically distinct and example, the polypeptide encoding the receptor, or a frag connect at distal capillary beds. ment thereof, can be supplied to an environment in order to 0003. The network of vessels that comprise the arterial and compete with cell-bound receptors, thereby effectively low venous systems develops by a process of directed movement ering or preventing activation of the cell-bound receptors. of endothelial tubes to desired cell masses and/or tissues. Also, the various methods of the present invention are useful During embryogenesis, the initial vascular framework is in studying and treating conditions related to angiogenesis, defined by the de novo formation of the dorsal aortae and Such as ischemia and tumor growth. cardinal veins. Mature circulatory networks are formed when 0009. The inventors have identified and sequenced the endothelial tubes sprout from central vessels, navigate gene that encodes the receptor, identified at least one ligand through the embryo, and reach their target cell mass and/or for the receptor (the slit ligand), and identified sequence and tissue. Upon reaching the target, the tubes are able to Supply structural similarities between the novel receptor and a family blood as nourishment for the tissue. of existing receptors—the Roundabout family of receptors. 0004. This navigation of endothelial tubes is important not 0010. Also, the inventors have identified a function for the only during embryonic development when the vasculature is Robo-4 receptor. The receptor, following interaction with the first forming, but also in all physiological processes that slit ligand, inhibits the migration of endothelial tubes. The include the introduction of a blood Supply to a cell mass repulsive cue provided by Robo-4/slit interaction contributes and/or tissue. These processes include various disease condi to the directed navigation of endothelial tubes by steering the tions that are sustainable only because of the introduction of tubes away from a location having the ligand, such as a cell a blood Supply to a cell mass. Such as the Survival of a Solid expressing the ligand. cancerous tumor. The continued growth of a Solid tumor 0011. The Robo-4 receptor is expressed on sprouting requires the presence of a blood Supply that nourishes the endothelial tubes that form the perineural vasculature beds. cells of the tumor mass. Angiogenesis, a physiological pro The neural tubes produce and secrete the slit ligand. This cess in which new blood vessels are formed and directed into enables the directed navigation of the endothelial tubes away a target cell mass and/or tissue, is a critical step in tumor from the neural tubes. As a result of this negative cue and development and Survival. Accordingly, methods and com likely in combination with attractive cues, the endothelial positions that are able to interfere with this process could be tubes, through slit-Robo-4 binding interactions that result in useful in preventing the growth and/or Survival of tumors. directed navigation, form a vasculature network around the 0005. Other disease conditions exist in which the blood developing central nervous system. This interaction with the Supply to a particular tissue is blocked or otherwise impeded, central nervous system leads to the close association between thereby diminishing the Supply of nutrients to that particular the nervous and vasculature systems that is evident in both tissue.
Load more

Recommended publications
  • Cloning and Functional Studies of a Novel Gene Aberrantly Expressed in RB-Deficient Embryos
    Developmental Biology 207, 62–75 (1999) Article ID dbio.1998.9141, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cloning and Functional Studies of a Novel Gene Aberrantly Expressed in RB-Deficient Embryos Shyng-Shiou F. Yuan,* Laura A. Cox,*,1 Gopal K. Dasika,* and Eva Y.-H. P. Lee*,2 *Department of Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245 The tumor suppressor RB regulates diverse cellular processes such as G1/S transition, cell differentiation, and cell survival. Indeed, Rb-knockout mice exhibit phenotypes including ectopic mitosis, defective differentiation, and extensive apoptosis in the neurons. Using differential display, a novel gene, Rig-1, was isolated based on its elevated expression in the hindbrain and spinal cord of Rb-knockout embryos. The longest open reading frame of Rig-1 encoded a polypeptide that consists of a putative extracellular segment with five immunoglobulin-like domains and three fibronectin III-like domains, a putative transmembrane domain, and a distinct intracellular segment. The Rig-1 sequence was 40% identical to the recently identified roundabout protein. Consistent with the predicted transmembrane nature of the protein, Rig-1 protein was present in the membranous fraction. Antisera raised against the putative extracellular and intracellular segments of Rig-1 reacted with an ;210-kDa protein in mouse embryonic CNS. Rig-1 mRNA was transiently expressed in the embryonic hindbrain and spinal cord. Elevated levels of Rig-1 mRNA and protein were found in Rb2/2 embryos.
    [Show full text]
  • Supplemental Information
    Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig.
    [Show full text]
  • Robo4 Is a Vascular-Specific Receptor That Inhibits
    Available online at www.sciencedirect.com R Developmental Biology 261 (2003) 251–267 www.elsevier.com/locate/ydbio Robo4 is a vascular-specific receptor that inhibits endothelial migration Kye Won Park,a,b Clayton M. Morrison,a Lise K. Sorensen,a Christopher A. Jones,a,b Yi Rao,c Chi-Bin Chien,d Jane Y. Wu,e Lisa D. Urness,f and Dean Y. Lia,b,f,* a Program in Human Molecular Biology and Genetics, School of Medicine, University of Utah, Salt Lake City, UT 84112, USA b Department of Oncological Sciences, School of Medicine, University of Utah, Salt Lake City, UT 84112, USA c Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110, USA d Department of Anatomy and Neurobiology, School of Medicine, University of Utah, Salt Lake City, UT 84112, USA e Departments of Pediatrics and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA f Division of Cardiology, School of Medicine, University of Utah, Salt Lake City, UT 84112, USA Received for publication 4 December 2002, revised 17 April 2003, accepted 23 April 2003 Abstract Guidance and patterning of axons are orchestrated by cell-surface receptors and ligands that provide directional cues. Interactions between the Robo receptor and Slit ligand families of proteins initiate signaling cascades that repel axonal outgrowth. Although the vascular and nervous systems grow as parallel networks, the mechanisms by which the vascular endothelial cells are guided to their appropriate positions remain obscure. We have identified a putative Robo homologue, Robo4, based on its differential expression in mutant mice with defects in vascular sprouting.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • Molecular Discrimination of Cutaneous Squamous Cell Carcinoma from Actinic Keratosis and Normal Skin
    Modern Pathology (2011) 24, 963–973 & 2011 USCAP, Inc. All rights reserved 0893-3952/11 $32.00 963 Molecular discrimination of cutaneous squamous cell carcinoma from actinic keratosis and normal skin Seong Hui Ra, Xinmin Li and Scott Binder Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA Actinic keratosis is widely believed to be a neoplastic lesion and a precursor to invasive squamous cell carcinoma. However, there has been some debate as to whether actinic keratosis is in fact actually squamous cell carcinoma and should be treated as such. As the clinical management and prognosis of patients is widely held to be different for each of these lesions, our goal was to identify unique gene signatures using DNA microarrays to discriminate among normal skin, actinic keratosis, and squamous cell carcinoma, and examine the molecular pathways of carcinogenesis involved in the progression from normal skin to squamous cell carcinoma. Formalin-fixed and paraffin-embedded blocks of skin: five normal skins (pooled), six actinic keratoses, and six squamous cell carcinomas were retrieved. The RNA was extracted and amplified. The labeled targets were hybridized to the Affymetrix human U133plus2.0 array and the acquisition and initial quantification of array images were performed using the GCOS (Affymetrix). The subsequent data analyses were performed using DNA-Chip Analyzer and Partek Genomic Suite 6.4. Significant differential gene expression (42 fold change, Po0.05) was seen with 382 differentially expressed genes between squamous cell carcinoma and normal skin, 423 differentially expressed genes between actinic keratosis and normal skin, and 9 differentially expressed genes between actinic keratosis and squamous cell carcinoma.
    [Show full text]
  • Genome-Wide Linkage Analysis of Human Auditory Cortical Activation Suggests Distinct Loci on Chromosomes 2, 3, and 8
    The Journal of Neuroscience, October 17, 2012 • 32(42):14511–14518 • 14511 Behavioral/Systems/Cognitive Genome-Wide Linkage Analysis of Human Auditory Cortical Activation Suggests Distinct Loci on Chromosomes 2, 3, and 8 Hanna Renvall,1* Elina Salmela,2,3* Minna Vihla,1 Mia Illman,1 Eira Leinonen,2,3 Juha Kere,2,3,4 and Riitta Salmelin1 1Brain Research Unit and MEG Core, O.V. Lounasmaa Laboratory, Aalto University, FI-00076 Aalto, Finland, 2Department of Medical Genetics, Haartman Institute, and Research Programs Unit, Molecular Medicine, University of Helsinki, FI-00014 Helsinki, Finland, 3Folkha¨lsan Institute of Genetics, FI-00014 Helsinki, Finland, and 4Department of Biosciences and Nutrition, and Science for Life Laboratory, Karolinska Institute, SE-14183 Stockholm, Sweden Neural processes are explored through macroscopic neuroimaging and microscopic molecular measures, but the two levels remain primarily detached. The identification of direct links between the levels would facilitate use of imaging signals as probes of genetic function and, vice versa, access to molecular correlates of imaging measures. Neuroimaging patterns have been mapped for a few isolated genes,chosenbasedontheirconnectionwithaclinicaldisorder.Hereweproposeanapproachthatallowsanunrestricteddiscoveryofthe genetic basis of a neuroimaging phenotype in the normal human brain. The essential components are a subject population that is composed of relatives and selection of a neuroimaging phenotype that is reproducible within an individual and similar between relatives but markedly variable across a population. Our present combined magnetoencephalography and genome-wide linkage study in 212 healthy siblings demonstrates that auditory cortical activation strength is highly heritable and, specifically in the right hemisphere, regulatedoligogenicallywithlinkagestochromosomes2q37,3p12,and8q24.TheidentifiedregionsdelimitascandidategenesTRAPPC9, operating in neuronal differentiation, and ROBO1, regulating projections of thalamocortical axons.
    [Show full text]
  • Anti-Robo4 Antibodies and Uses Therefor
    (19) TZZ T (11) EP 2 468 776 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 27.06.2012 Bulletin 2012/26 C07K 16/30 (2006.01) A61K 39/395 (2006.01) A61P 35/00 (2006.01) C07K 16/28 (2006.01) (21) Application number: 11195237.0 (22) Date of filing: 08.02.2008 (84) Designated Contracting States: • Koch, Alexander W. AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Millbrae, CA 94030 (US) HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT • Wu, Yan RO SE SI SK TR Foster City, CA 94404 (US) Designated Extension States: • Stawicki, Scott AL BA MK RS San Francisco, CA 94105 (US) • Carano, Richard (30) Priority: 09.02.2007 US 88921407 P San Ramon, CA 94583 (US) 23.02.2007 US 89147507 P (74) Representative: Woolley, Lindsey Claire et al (62) Document number(s) of the earlier application(s) in Mewburn Ellis LLP accordance with Art. 76 EPC: 33 Gutter Lane 08729348.6 / 2 125 896 London EC2V 8AS (GB) (71) Applicant: Genentech, Inc. South San Francisco, CA 94080 (US) Remarks: •This application was filed on 22-12-2011 as a (72) Inventors: divisional application to the application mentioned • Peale, Jr. Franklin V. under INID code 62. San Carlos, CA 94070 (US) •Claims filed after the date of filing of the application • Watts, Ryan J. / after the date of receipt of the divisional application San Mateo, CA 94402 (US) (Rule 68(4) EPC). (54) Anti-Robo4 antibodies and uses therefor (57) The invention provides anti-Robo4 antibodies, ods of using these antibodies, including diagnostic and and compositions comprising the antibodies and meth- therapeutic methods.
    [Show full text]
  • USP33 Mediates Slitrobo Signaling in Inhibiting Colorectal Cancer Cell
    IJC International Journal of Cancer USP33 mediates Slit-Robo signaling in inhibiting colorectal cancer cell migration Zhaohui Huang1,2, Pushuai Wen3, Ruirui Kong3, Haipeng Cheng2, Binbin Zhang1, Cao Quan1, Zehua Bian1, Mengmeng Chen3, Zhenfeng Zhang4, Xiaoping Chen2, Xiang Du5, Jianghong Liu3, Li Zhu3, Kazuo Fushimi2, Dong Hua1 and Jane Y. Wu2,3 1 Wuxi Oncology Institute, the Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, China 2 Department of Neurology, Center for Genetic Medicine, Lurie Cancer Center, Northwestern University Feinberg School of Medicine, 303 E. Chicago Ave., Chicago, IL 3 State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 4 State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, China 5 Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, China Originally discovered in neuronal guidance, the Slit-Robo pathway is emerging as an important player in human cancers. How- ever, its involvement and mechanism in colorectal cancer (CRC) remains to be elucidated. Here, we report that Slit2 expression is reduced in CRC tissues compared with adjacent noncancerous tissues. Extensive promoter hypermethylation of the Slit2 gene has been observed in CRC cells, which provides a mechanistic explanation for the Slit2 downregulation in CRC. Func- tional studies showed that Slit2 inhibits CRC cell migration in a Robo-dependent manner. Robo-interacting ubiquitin-specific protease 33 (USP33) is required for the inhibitory function of Slit2 on CRC cell migration by deubiquitinating and stabilizing Robo1. USP33 expression is downregulated in CRC samples, and reduced USP33 mRNA levels are correlated with increased tumor grade, lymph node metastasis and poor patient survival.
    [Show full text]
  • Mir‑218 Functions As a Tumor Suppressor Gene in Cervical Cancer
    MOLECULAR MEDICINE REPORTS 21: 209-219, 2020 miR‑218 functions as a tumor suppressor gene in cervical cancer ZHEN LIU1, LIN MAO2, LINLIN WANG1, HONG ZHANG2 and XIAOXIA HU1 1Department of Gynecology, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi 530021; 2Department of Gynecology, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, P.R. China Received October 23, 2018; Accepted April 12, 2019 DOI: 10.3892/mmr.2019.10809 Abstract. Previous microRNA (miR) microarray analysis in regulating cell proliferation, adhesion and migration, and revealed that miR‑218 is downregulated in cervical cancer the cell cycle. In conclusion, the findings of the present study tissues. The present study aimed to further evaluate the suggested that miR‑218 may possess antitumor activities in expression of miR‑218 in cervical cancer specimens, deter- cervical cancer. mine the association between its expression with disease progression, and investigate the roles of miR‑218 in cervical Introduction cancer cells. Tissue specimens were obtained from 80 patients with cervical squamous cell carcinoma, 30 patients with Cervical cancer remains a significant global health problem, high-grade cervical intraepithelial neoplasia [(CIN) II/III] and particularly in developing countries, where cervical cancer is 15 patients with low-grade CIN (CINI); in addition, 60 plasma a leading cause of cancer-associated death in women (1). The samples were obtained from patients with cervical cancer, and risk factors for cervical cancer development primarily include 15 normal cervical tissue specimens and 30 plasma samples human papillomavirus (HPV) infection (accounting for ~90% were obtained from healthy women. These samples were used of cervical cancer cases), tobacco smoking, long-term use of for analysis of miR‑218 expression via reverse transcription‑­ oral contraceptives, multiple pregnancies, and genetic and quantitative PCR.
    [Show full text]
  • In Vivo Functional Analysis of Drosophila Robo1 Fibronectin Type-III Repeats
    INVESTIGATION In Vivo Functional Analysis of Drosophila Robo1 Fibronectin Type-III Repeats Haley E. Brown, Marie C. Reichert,1 and Timothy A. Evans2 Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas 72701 ORCID ID: 0000-0002-2756-8064 (T.A.E.) ABSTRACT The repellant ligand Slit and its Roundabout (Robo) family receptors regulate midline crossing of KEYWORDS axons during development of the embryonic central nervous system (CNS). Slit proteins are produced at the Drosophila midline and signal through Robo receptors to repel axons from the midline. Disruption of Slit-Robo signaling Slit causes ectopic midline-crossing phenotypes in the CNS of a broad range of animals, including insects and Robo vertebrates. While previous studies have investigated the roles of Drosophila melanogaster Robo1’s five axon guidance Immunoglobulin-like (Ig) domains, little is known about the importance of the three evolutionarily conserved midline crossing Fibronectin (Fn) type-III repeats. We have individually deleted each of Drosophila Robo1’s three Fn repeats, fibronectin type-III and then tested these Robo1 variants in vitro to determine their ability to bind Slit in cultured Drosophila cells repeat and in vivo to investigate the requirement for each domain in regulating Robo1’sembryonicexpression pattern, axonal localization, midline repulsive function, and sensitivity to Commissureless (Comm) downregu- lation. We demonstrate that the Fn repeats are not required for Robo1 to bind Slit or for proper expression of Robo1 in Drosophila embryonic neurons. When expressed in a robo1 mutant background, these variants are able to restore midline repulsion to an extent equivalent to full-length Robo1. We identify a novel requirement for Fn3 in the exclusion of Robo1 from commissures and downregulation of Robo1 by Comm.
    [Show full text]
  • The Functional and Structural Analysis of Drosophila Robo2 in Axon Guidance
    University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 8-2019 The Functional and Structural Analysis of Drosophila robo2 in Axon Guidance LaFreda Janae Howard University of Arkansas, Fayetteville Follow this and additional works at: https://scholarworks.uark.edu/etd Part of the Cell Biology Commons, Computational Biology Commons, Genetics Commons, and the Molecular and Cellular Neuroscience Commons Recommended Citation Howard, LaFreda Janae, "The Functional and Structural Analysis of Drosophila robo2 in Axon Guidance" (2019). Theses and Dissertations. 3379. https://scholarworks.uark.edu/etd/3379 This Dissertation is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected]. The Functional and Structural Analysis of Drosophila robo2 in Axon Guidance A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cell and Molecular Biology by LaFreda Janae Howard Fort Valley State University Bachelor of Science in Biology, 2014 August 2019 University of Arkansas This dissertation is approved for recommendation to the Graduate Council. Timothy A. Evans, Ph.D. Dissertation Advisor Jeffrey Lewis, Ph.D. Ines Pinto, Ph.D. Committee Member Committee Member Michael Lehmann, Ph.D. Paul Adams, Ph.D. Committee Member Committee Member ABSTRACT In animals with complex nervous systems such as mammals and insects, signaling pathways are responsible for guiding axons to their appropriate synaptic targets. Importantly, when this process is not successful during the development of an organism, outcomes include catastrophes such as human neurological diseases and disorders.
    [Show full text]
  • Investigation of the Regulation of Roundabout4 by Hypoxia-Inducible Factor-1A in Microvascular Endothelial Cells
    Retina Investigation of the Regulation of Roundabout4 by Hypoxia-Inducible Factor-1a in Microvascular Endothelial Cells Rui Tian,1 Zaoxia Liu,1 Hui Zhang,1 Xuexun Fang,2 Chenguang Wang,1 Shounan Qi,1 Yan Cheng,1 and Guanfang Su1 1Department of Ophthalmology, Second Hospital of Jilin University, Changchun, Jilin, China 2School of Life Science, Jilin University, Changchun, China Correspondence: Guanfang Su, De- PURPOSE. We determined if hypoxia-inducible factor-1a (HIF-1a) and Roundabout4 (Robo4) partment of Ophthalmology, Second colocalized in fibrovascular membranes (FVM) from patients with proliferative diabetic Hospital of Jilin University, 218 retinopathy (PDR), and investigated the regulation of HIF-1a on Robo4 in microvascular Ziqiang Street, Changchun, Jilin, endothelial cells under normoxic and hypoxic conditions in vitro. 130021, China; [email protected]. METHODS. Immunofluorescence and confocal laser scanning microscopy were done to analyze Submitted: March 21, 2014 the colocalization of HIF-1a and Robo4 in the FVM. Expression of HIF-1a was knocked down Accepted: February 27, 2015 by small interfering RNA (siRNA) technology to study its effects on Robo4 expression of human retinal endothelial cells (HREC) and human dermal microvascular endothelial cells Citation: Tian R, Liu Z, Zhang H, et al. (HDMEC) under normoxic and/or hypoxic conditions. Full-length human a gene was Investigation of the regulation of HIF-1 Roundabout4 by hypoxia-inducible transfected into HREC and HDMEC using GFP lentivirus vectors to overexpress HIF-1a under factor-1a in microvascular endothelial normoxic conditions. The HIF-1a and Robo4 mRNA and protein expressions were quantified cells. Invest Ophthalmol Vis Sci. by real-time PCR and Western blot.
    [Show full text]