转化生长因子-β激活的长链非编码RNA通过上皮-间充质转化调控人膀胱癌细胞生物学行为的机制

The mechanism of long non-coding RNA activated by transforming growth factor-βregulating the biological behavior of human bladder cancer cells by epithelial-mesenchymal transition

目的探讨转化生长因子-β激活的长链非编码RNA(lncRNA-ATB)与膀胱尿路上皮癌上皮-间充质转化(EMT)的相关性及lncRNA-ATB对膀胱癌细胞生物学行为的影响。方法应用实时定量反转录聚合酶链反应(RT-qPCR)法检测lncRNA-ATB在人正常尿路上皮细胞株SV-HUC-1和膀胱癌细胞株EJ中的表达,在SV-HUC-1细胞通过转染pcDNA3.1-ATB过表达lncRNA-ATB,在EJ细胞通过转染sh-ATB敲低lncRNA-ATB,免疫荧光检测细胞形态学变化,RT-qPCR法和蛋白质印迹法(Western blot)分别检测EMT相关基因mRNA及蛋白水平表达变化,细胞计数试剂盒(CCK-8)检测细胞的增殖能力,Transwell检测细胞的侵袭能力。两组间比较采用独立样本t检验。结果lncRNA-ATB在EJ细胞株的表达明显高于SV-HUC-1细胞株(2.836±0.317比0.981±0.234,t=-14.109,P<0.05),SV-HUC-1细胞转染pcDNA3.1-ATB后,细胞由卵圆形转变为梭形(EMT表型变化)。pcDNA3.1-ATB转染组细胞EMT相关基因E盒结合锌指蛋白1(ZEB1)和E盒结合锌指蛋白2(ZEB2)mRNA和蛋白表达均高于对照组(pcDNA3.1-NC)(3.682±0.270比0.969±0.119、4.119±0.216比0.970±0.156;1.439±0.151比0.209±0.012、1.057±0.993比0.239±0.025,t=-27.543、-35.503、-14.074、-13.843,P<0.05),E-钙黏蛋白(E-cadherin)mRNA和蛋白表达均低于对照组(0.383±0.023比0.985±0.182、0.270±0.035比0.540±0.104,t=9.826、4.269,P<0.05),细胞增殖能力增强(48 h:0.548±0.041比0.348±0.035、72 h:0.906±0.118比0.468±0.039、96 h:1.432±0.224比0.731±0.078,t=-11.081、-10.591、-8.866,P<0.05),细胞侵袭能力增强(293.111±26.770比69.556±7.939,t=-24.019,P<0.05)。EJ细胞转染sh-ATB后,细胞由梭形转变为卵圆形,与对照组sh-NC比较,ZEB1和ZEB2 mRNA和蛋白表达下降(0.385±0.059比0.950±0.181、0.341±0.051比0.992±0.175;0.244±0.154比0.764±0.049、0.067±0.012比0.449±0.197,t=8.907、10.713、17.658、28.844,P<0.05),E-cadherin mRNA和蛋白表达增高(3.697±0.289比0.929±0.212、0.377±0.29比0.132±0.014,t=-23.151、-10.921,P<0.05),细胞增殖能力下降(72 h:0.734±0.063比0.973±0.161、96 h:0.926±0.130比1.520±0.053,t=4.139、12.752,P<0.05),细胞侵袭能力降低(81.222±8.273比335.000±16.875,t=40.511,P<0.05)。结论lncRNA-ATB可能通过上调ZEB1和ZEB2表达进而下调E-cadherin表达诱导细胞EMT变化促进膀胱癌增殖和侵袭。

Objective To explore the correlation between long non-coding RNA activated by transforming growth factor-β(lncRNA-ATB)and epithelial mesenchymal transition(EMT)in bladder urothelial carcinoma and the effect of lncRNA ATB on the biological behavior of bladder cancer cells.Methods Real-time PCR was used to detect the expression of lncRNA-ATB in human normal urothelial cell line SV-HUC-1 and bladder cancer cell line EJ.SV-HUC-1 and EJ cells were transfected with pcDNA3.1-ATB and sh-ATB separately.Immunofluorescence was used to detect the cell morphology changes.The mRNA and protein levels of EMT related genes were detected by Real-time PCR and Western blotting in cells.The proliferation of cells was detected by cell counting kit-8(CCK-8)assay.The invasive ability of cells was assessed by Transwell assay.T-test was used for comparison between two groups.Results The expression of lncRNA-ATB in EJ cell line was significantly higher than that in SV-HUC-1 cell line(t=-14.109,P<0.05).After transfected with pcDNA3.1-ATB in SV-HUC-1 cells,the cells transformed from oval to spindle shape(EMT-like changes).We also found that overexpression of lncRNA-ATB leaded to increased expression of mesenchymal markers zinc finger E-box binding protein 1(ZEB1)and zinc finger E-box binding protein 2(ZEB2)(3.682±0.270 vs.0.969±0.119,4.119±0.216 vs.0.970±0.156;1.439±0.151 vs.0.209±0.012,1.057±0.993 vs.0.239±0.025,t=-27.543,-35.503,-14.074,-13.843,P<0.05),and decreased expression of epithelial markers E-cadherin(0.383±0.023 vs.0.985±0.182,0.270±0.035 vs.0.540±0.104,t=9.826,4.269,P<0.05)at mRNA and protein levels and significantly promoted cell proliferation capability(48 h:0.548±0.041 vs.0.348±0.035,72 h:0.906±0.118 vs.0.468±0.039,96 h:1.432±0.224 vs.0.731±0.078,t=-11.081,-10.591,-8.866,P<0.05)and invasion capability(293.111±26.770 vs.69.556±7.939,t=-24.019,P<0.05)in vitro.After transfected with sh-ATB in EJ cells,the cells transformed from spindle to oval shape.Knockdown of lncRNA-ATB resulted in decreased expression of mesenchymal markers ZEB1 and ZEB2(0.385±0.059 vs.0.950±0.181,0.341±0.051 vs.0.992±0.175;0.244±0.154 vs.0.764±0.049,0.067±0.012 vs.0.449±0.197,t=8.907,10.713,17.658,28.844,P<0.05)and increased expression of E-cadherin(3.697±0.289 vs.0.929±0.212,0.377±0.29 vs.0.132±0.014,t=-23.151,-10.921,P<0.05)in EJ cells at mRNA and protein levels and reduced their capability of proliferation(72 h:0.734±0.063 vs.0.973±0.161,96 h:0.926±0.130 vs.1.520±0.053,t=4.139,12.752,P<0.05)and invasion(81.222±8.273 vs.335.000±16.875,t=40.511,P<0.05)in vitro.Conclusion Our results suggest that lncRNA-ATB may plays a crucial role in the proliferation and invasion of bladder cancer cell by promoting EMT process by suppressing E-cadherin expression through up regulating the expression of ZEB1 and ZEB2.