长链非编码RNA CCAT1通过负调控miR-218-5p促进人视网膜母细胞瘤SO-RB50和Y79细胞的生长。
Long non-coding RNA CCAT1 promotes human retinoblastoma SO-RB50 and Y79 cells through negative regulation of miR-218-5p.
作者信息
Zhang Hongxu, Zhong Jianguang, Bian Zhenyu, Fang Xiang, Peng You, Hu Yongping
机构信息
Department of Ophtalmology, Hangzhou First People's Hospital, Nanjing Medical University, Hangzhou 310006, Zhejiang, PR China.
Department of Orthopaedics, Hangzhou First People's Hospital, Nanjing Medical University, Hangzhou Orthopedic Institute, Hangzhou 310006, Zhejiang, PR China.
出版信息
Biomed Pharmacother. 2017 Mar;87:683-691. doi: 10.1016/j.biopha.2017.01.004. Epub 2017 Jan 12.
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).
CONCLUSION
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.
目的
探讨长链非编码RNA（lncRNA）在人视网膜母细胞瘤（RB）中的调控作用及潜在机制。
方法
通过基因芯片和定量逆转录PCR（qRT-PCR）分析RB组织中的lncRNA谱。选择其中一种鉴定出的lncRNA（LncRNA CCAT1）进行进一步实验。分别用阴性对照、靶向lncRNA CCAT1的小干扰RNA（si-CCAT1）和si-CCAT1+miR218-5p抑制剂转染SO-RB50和Y79细胞。通过qRT-PCR检测lncRNA CCAT1的表达。分别用CCK8、划痕实验和Transwell实验检测细胞增殖、迁移和侵袭能力。通过流式细胞术评估细胞凋亡和细胞周期分布。通过蛋白质免疫印迹法分析凋亡相关蛋白（cle-caspase-3、cle-caspase-9、Bax和Bcl-2）和细胞周期相关蛋白表达（细胞周期蛋白B1、细胞周期蛋白依赖性激酶2（CDC2）和磷酸化CDC2（Thr161））。
结果
转染si-CCAT1后，SO-RB50和Y79细胞中lncRNA CCAT1的表达显著受到抑制（P<0.01）。两种RB细胞在转染si-CCAT1后48小时均表现出增殖、迁移和侵袭能力显著降低，但凋亡明显增加（P<0.05或0.01）。在转染后24和48小时，si-CCAT1+miR218-5p抑制剂组的RB细胞增殖、迁移和侵袭能力明显高于si-CCAT1组，但凋亡明显低于si-CCAT1组（均P<0.05或0.01）。si-CCAT1显著增加cle-caspase-3、cle-caspase-9、Bax的表达，但降低Bcl-2的表达（P<0.01）。与阴性对照组相比，siCCAT1组中SO-RB50和Y79细胞的G2/M期比例显著增加（P<0.01）。lncRNA CCAT1干扰显著降低细胞周期蛋白B1、CDC2和磷酸化CDC2（Thr161）的表达（P<0.01）。
结论
LncRNA CCAT1可能通过负向调节miR-218-5p促进SO-RB50和Y79细胞的增殖、迁移和侵袭，并减少细胞凋亡。我们的研究表明lncRNA CCAT1可能是RB的潜在生物标志物和治疗靶点。
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