title: Oncogenic microRNA 17-92 cluster is regulated by epithelial cell adhesion molecule and could be a potential therapeutic target in retinoblastoma.
abstract: OBJECTIVE
  Several miRNAs have been reported as candidate oncogenes and tumor suppressors, which are involved in the pathways specifically altered during tumorigenesis or metastasis. The miR 17-92 cluster located in 13q31 locus might contribute to retinoblastoma (RB) oncogenesis as 13q31 is amplified often in RB. We attempted to identify the factors involved in the regulation of miR 17-92 cluster in RB.
  
  
  METHODS
  Real-time quantitative reverse transcriptase PCR was performed to study the expression of the miR 17-92 cluster in primary RB tumors and in Y79 cells after epithelial cell adhesion molecule (EpCAM) silencing. EpCAM was silenced using siRNA and confirmed by western blotting. The Y79 cells were transfected with individual and mixed antagomirs and studied the cell viability by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, invasion by matrigel analysis and caspase-3 expression by flow cytometry.
  
  
  RESULTS
  The relative expression of miR 17-92 cluster, compared to that of a normal retina, ranged from 25 to 220 fold (p<0.0001), miR-18 being highly expressed in RB. Post EpCAM silencing resulted in a significant decrease (p<0.01) in the expression of the miR 17-92 cluster by 4 to eightfold in Y79 cells. Y79 cells transfected with an antagomirs mix (all 5 miRNAs) showed decreased cell viability (p<0.001) and cell invasion (p<0.001). Similarly, Y79 cells treated with antagomirs mix showed increased expression of caspase-3 (p<0.001), which confirms the anti-proliferative effect of antagomirs.
  
  
  CONCLUSIONS
  This study has showed varied expression of the miR17-92 cluster in primary RB tumors. EpCAM influences miR 17-92 cluster expression in retinoblastoma. In addition, we showed that the miR 17-92 cluster plays a role in RB cell proliferation and invasion. Therefore, targeting the miRNA 17-92 cluster may be beneficial for controlling Y79/RB cell proliferation and invasion.
authors.affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.
authors.forename: Moutushy Mitra
authors.initials: MM
authors.lastname: Kandalam
authors.forename: Madhu
authors.initials: M
authors.lastname: Beta
authors.forename: Uma K
authors.initials: UK
authors.lastname: Maheswari
authors.forename: S
authors.initials: S
authors.lastname: Swaminathan
authors.forename: Subramanian
authors.initials: S
authors.lastname: Krishnakumar
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics* / Q000378:metabolism; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D002648:Child; D002675:Child, Preschool; D002882:Chromosomes, Human, Pair 13; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007223:Infant; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics; D062085:RNA, Long Noncoding; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D019572:Retinal Neoplasms / Q000235:genetics* / Q000378:metabolism; D012175:Retinoblastoma / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; C568997:MIR17HG, human; D035683:MicroRNAs; D009841:Oligonucleotides; D062085:RNA, Long Noncoding; D034741:RNA, Small Interfering; D053148:Caspase 3