title: Long non-coding RNA CCAT1 promotes human retinoblastoma SO-RB50 and Y79 cells through negative regulation of miR-218-5p.
abstract: OBJECTIVE
  To investigate the regulatory role and potential mechanism of long non-coding RNAs (lncRNA) in human retinoblastoma (RB).
  
  
  METHODS
  The lncRNA profile in RB tissues were analyzed by microarray and quantitative reverse transcription PCR (qRT-PCR). One of the identified lncRNAs (LncRNA CCAT1) was selected for further experiments. SO-RB50 and Y79 cells were transfected with negative control, siRNA targeting lncRNA CCAT1 (si-CCAT1) and si-CCAT1+miR218-5p inhibitor, respectively. lncRNA CCAT1 expression was measured by qRT-PCR. Cell proliferation, migration and invasion were detected by CCK8, wound scratching, and transwell assay, respectively. Apoptosis and cell cycle distribution were assessed by flow cytometry. Apoptosis- (cle-caspase-3, cle-caspase-9, Bax and Bcl-2) and cell cycle-related protein expression (cyclin B1, CDC2 and p-CDC2 (Thr161)) were analyzed by Western blot.
  
  
  RESULTS
  lncRNA CCAT1 expression in SO-RB50 and Y79 cells was significantly inhibited after si-CCAT1 transfection (P<0.01). Both RB cells exhibited significantly reduced proliferation, migration and invasion abilities, but markedly increased apoptosis at 48h after si-CCAT1 transfection (P<0.05 or 0.01). RB cells in si-CCAT1+miR218-5p inhibitor group had significantly higher proliferation, migration and invasion, but notably lower apoptosis compared with si-CCAT1 group at 24 and 48h after transfection (all P<0.05 or 0.01). si-CCAT1 significantly increased the expression of cle-caspase-3, cle-caspase-9, Bax, but decreased Bcl-2 expression (P<0.01). The proportion of G2/M SO-RB50 and Y79 cells in siCCAT1 group was significantly increased compared with negative control group (P<0.01). LncRNA CCAT1 interference significantly reduced the expression of cyclin B1, CDC2 and p-CDC2 (Thr161) (P<0.01).
  
  
  CONCLUSIONS
  LncRNA CCAT1 promotes the proliferation migration and invasion, and reduces cell apoptosis of SO-RB50 and Y79 cells, probably through negative modulation of miR-218-5p. Our study suggested lncRNA CCAT1 as a potential biomarker and therapeutic target for RB.
authors.affiliation: Department of Ophtalmology, Hangzhou First People's Hospital, Nanjing Medical University, Hangzhou 310006, Zhejiang, PR China.
authors.forename: Hongxu
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Ophtalmology, Hangzhou First People's Hospital, Nanjing Medical University, Hangzhou 310006, Zhejiang, PR China.
authors.forename: Jianguang
authors.initials: J
authors.lastname: Zhong
authors.affiliation: Department of Orthopaedics, Hangzhou First People's Hospital, Nanjing Medical University, Hangzhou Orthopedic Institute, Hangzhou 310006, Zhejiang, PR China.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Bian
authors.affiliation: Department of Central Laboratory, Hangzhou First People's Hospital, Nanjing Medical University, Hangzhou 310006, Zhejiang, PR China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Fang
authors.affiliation: Department of Surgical Oncology, Hangzhou First People's Hospital, Nanjing Medical University, Hangzhou 310006, Zhejiang, PR China.
authors.forename: You
authors.initials: Y
authors.lastname: Peng
authors.affiliation: Department of Ophtalmology, Hangzhou First People's Hospital, Nanjing Medical University, Hangzhou 310006, Zhejiang, PR China. Electronic address: yoephu@yeah.net.
authors.forename: Yongping
authors.initials: Y
authors.lastname: Hu
mesh_terms: D017209:Apoptosis / Q000235:genetics; D053148:Caspase 3 / Q000235:genetics; D053453:Caspase 9 / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D016195:G2 Phase / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D062085:RNA, Long Noncoding / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D012175:Retinoblastoma / Q000235:genetics* / Q000473:pathology; D014162:Transfection / Q000379:methods; D051028:bcl-2-Associated X Protein / Q000235:genetics
chemical_list: C579842:CCAT1 long noncoding RNA, human; C547471:MIRN218 microRNA, human; D035683:MicroRNAs; D019253:Proto-Oncogene Proteins c-bcl-2; D062085:RNA, Long Noncoding; D034741:RNA, Small Interfering; D051028:bcl-2-Associated X Protein; D053148:Caspase 3; D053453:Caspase 9
keywords: Apoptosis; Cell cycle; LncRNACCAT1; Metastasis; Retinoblastoma