八聚体结合转录因子4假基因5对膀胱癌T24细胞迁移和侵袭行为的影响

The effect of octamer-binding transcription factor 4 pseudogene 5 on bladder cancer cell line T24 migration and invasion

目的探讨八聚体结合转录因子4假基因5(OCT4-pg5)对人膀胱癌T24细胞迁移和侵袭的影响及其机制。方法采用实时定量反转录聚合酶链反应(RT-qPCR)检测OCT4-pg5在5种膀胱癌细胞株和膀胱永生化上皮细胞SV-HUC-1中的表达。利用Lipfectamine 2000将OCT4-pg5干扰质粒瞬时转染至膀胱癌T24细胞中,通过RT-qPCR检测各组细胞中OCT4-pg5的转染效果;通过细胞划痕实验和Transwell侵袭实验检测干扰OCT4-pg5的表达对T24细胞迁移和侵袭的影响;RT-qPCR及蛋白质印迹法(Western blot)检测上皮型钙黏附蛋白(E-cadherin)、波形蛋白(Vimentin)、β-连环蛋白(β-catenin)表达变化。结果与膀胱永生化上皮细胞(SV-HUC-1)比较,OCT4-pg5在膀胱癌细胞(5637、EJ、BIU-87、TCCSUP和T24)中的表达增加,以T24细胞最为明显(P<0.01)。RT-qPCR结果显示:转染si-OCT4-pg5干扰质粒后,T24细胞中OCT4-pg5表达明显降低(P<0.01)。划痕实验及Transwell侵袭实验结果表明,干扰OCT4-pg5表达后,T24细胞的迁移率为(28.50±4.08)%,细胞穿过膜数目为(104.50±6.95)个;分别与相应阴性对照组比较明显减弱[(43.26±5.49)%,P<0.01;(140.50±8.90)个,P<0.01]。RT-qPCR和Western blot结果显示:与阴性对照组比较,干扰OCT4-pg5表达后,T24细胞的E-cadherin表达增加(P<0.01),Vimentin和β-catenin表达减少(P<0.01,P<0.01)。结论OCT4-pg5在膀胱癌细胞中呈高表达,干扰OCT4-pg5表达能抑制膀胱癌T24细胞迁移和侵袭,其机制可能与其影响E-cadherin、Vimentin、β-catenin蛋白表达有关。

Objective To investigate octamer-binding transcription factor 4 pseudogene 5(OCT4-pg5)expression in bladder cancer cell lines and its effect on T24 migration and invasion and the potential mechanism.Methods The real-time quantitative reverse transeriptase-polymerase chain reaction(RT-qPCR)was conducted to detect OCT4-pg5 expression in 5 kinds of bladder cancer cell lines(5637,EJ,BIU-87,T24,TCCSUP)and one normal Bladder epithelial cell line SV-HUC-1.Si-OCT4-pg5 vector and its control vector were transfected into T24 cells by Lipofectamine 2000,and OCT4-pg5 expression was detected by RT-qPCR.Next,wound healing assay and Transwell assay were used to examine the effects of OCT4-pg5 on T24 cell migration and invasion.Moreover,the possible mechanism by which OCT4-pg5 mediated T24 migration and invasion was explored using Western blotting and RT-qPCR.Results In comparison to SV-HUC-1,OCT4-pg5 levels were up-regulated in bladder cancer cell lines,especially in T24 cell(P<0.01).RT-qPCR results showed that OCT4-pg5 espression was obviously down-regulated after being translated with si-OCT4-pg5 vector.OCT4-pg5 down-expression in T24 cell could inhibit cell migration[(28.50±4.08)%and(43.26±5.49)%,P<0.01]and invasion[(104.50±6.95)and(140.50±8.90)cells,P<0.01].In addition,down-regulation of OCT4-pg5 significantly reduced Vimentin(0.60±0.06)andβ-catenin(0.70±0.08)mRNA expression,and improved E-cadherin mRNA expression(1.50±0.10)when compared with their contral groups(1.00±0.02,1.00±0.04 and 1.00±0.05,respectively,P<0.01).Moreover,the same results were also obtained by western blotting.Conclusion OCT4-pg5 is overexpression in bladder cancer cells.And OCT4-pg5 down-expression in T24 cell could inhibit cell migration and invasion.OCT4-pg5 mediated T24 migration and invasion probably induced by regulating E-cadherin,Vimentin,β-catenin expression.