Shang Chao, Zhang Hui, Guo Yan, Hong Yang, Liu Yunhui, Xue Yixue
Department of Neurobiology, China Medical University, Shenyang, 110001, People's Republic of China.
Mol Biol Rep. 2014;41(4):2521-7. doi: 10.1007/s11033-014-3110-0. Epub 2014 Jan 18.
To investigate whether the miR-320a could regulate bladder cancer cells invasion by down-regulation of ITGB3. Real-time quantitative PCR was applied to evaluate the expression level of miRNA-320a in bladder transitional cell carcinomas (TCC) and normal bladder transitional cell (NBTC) samples. The invasion ability of miR-320a in TCC T24 cells was analyzed by Transwell assay after pre-miR-320a or anti-miR-125b transfection. For the invasion mechanism analysis of miR-320a on T24 cells, TargetScan, PicTar and miRBase were used to predict the possible target gene of miR-320a. Luciferase activities assay and western blot were used to reveal the predicted target gene of miR-320a were direct and specific. RNA interference technology was used to confirm the invasion inhibition of miR-320a was directly induced by ITGB3. Our study showed that miR-320a was down-regulated in human TCC specimens compared to that in NBTC specimens. Over-expression of miR-320a in T24 cells inhibited TCC invasion and this inhibitory effect on T24 cells could be restore by miR-320a knocked down. Mechanism analysis revealed that ITGB3 was a direct and specific target of miR-320a. The advanced effect of anti-miR-320a on TCC cell invasion was mediated by expression silence of ITGB3. In summary, aberrantly expressed miR-320a contribute to T24 cells invasion partly through directly down-regulating ITGB3 protein expression in TCC and this miRNA signature offers a novel potential therapeutic strategy for TCC.
为研究miR-320a是否可通过下调整合素β3(ITGB3)来调节膀胱癌细胞的侵袭。应用实时定量聚合酶链反应(PCR)评估miRNA-320a在膀胱移行细胞癌(TCC)和正常膀胱移行细胞(NBTC)样本中的表达水平。在转染前体miR-320a或抗miR-125b后,通过Transwell实验分析miR-320a对TCC T24细胞侵袭能力的影响。为分析miR-320a对T24细胞的侵袭机制,使用TargetScan、PicTar和miRBase预测miR-320a可能的靶基因。通过荧光素酶活性测定和蛋白质免疫印迹法(western blot)揭示miR-320a预测的靶基因是直接且特异的。采用RNA干扰技术证实miR-320a对侵袭的抑制作用是由ITGB3直接诱导的。我们的研究表明,与NBTC标本相比,miR-320a在人类TCC标本中表达下调。T24细胞中miR-320a的过表达抑制了TCC的侵袭,而这种对T24细胞的抑制作用可通过敲低miR-320a来恢复。机制分析显示,ITGB3是miR-320a直接且特异的靶标。抗miR-320a对TCC细胞侵袭的促进作用是由ITGB3的表达沉默介导的。总之,异常表达的miR-320a部分通过直接下调TCC中ITGB3蛋白的表达促进T24细胞的侵袭,并且这种微小RNA特征为TCC提供了一种新的潜在治疗策略。