干扰ORAI1表达对前列腺癌PC-3细胞迁移侵袭及上皮间质转化的影响

Effects of ORAI1 gene on cell migration and invasion and epithelial-mesenchymal transition in prostate cancer PC-3 cells

目的观察钙释放激活钙通道蛋白1(ORAI1)对前列腺癌PC-3细胞侵袭转移能力的影响并探讨其在上皮间质转化(EMT)过程中的作用。方法将携带ORAI1基因的小干扰RNA(shRNA)慢病毒载体LV-ORAI1-RNAi转染人前列腺癌PC-3细胞(KD组),同时设对照组(NC组),并添加转化生长因子β1(TGF-β1)。实时定量反转录聚合酶链反应(RT-qPCR)及Western blot验证抑制ORAI1表达的有效性。采用Celigo划痕实验及Transwell迁移、侵袭实验检测抑制ORAI1对细胞运动及迁移、侵袭能力的影响。采用Western blot检测EMT标志物E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波动蛋白(Vimentin)及smad3、p-smad3在各组细胞中含量的差异。结果与对照组相比,转染LV-ORAI1-RNAi后,细胞内ORAI1的mRNA及蛋白水平均降低。Celigo划痕实验显示,与NC组(14.38%±0.77%)比较,KD组(10.24%±1.17%)迁移率降低(P<0.05);与NC+TGF-β1组(26.21%±2.23%)比较,KD+TGF-β1组(12.44%±1.29%)迁移率降低(P<0.05)。Transwell迁移实验显示,与NC组(298±14.2)比较,KD组(229±8.4)迁移细胞数目降低(P<0.05);与NC+TGF-β1组(338±12.9)比较,KD+TGF-β1组(216±12.2)迁移细胞数目降低(P<0.05)。Transwell侵袭实验显示,与NC组(79±4.6)比较,KD组(46±3.4)侵袭细胞数目降低(P<0.05);与NC+TGF-β1组(123±5.1)比较,KD+TGF-β1组(66±5.5)侵袭细胞数目降低(P<0.05)。Western blot结果显示,与NC组比较,NC+TGF-β1组E-cadherin表达下降,N-cadherin和Vimentin表达升高;与NC+TGF-β1组比较,KD+TGF-β1组E-cadherin表达水平升高,N-cadherin、Vimentin和p-smad3表达下降。结论降低ORAI1表达后,PC-3细胞转移能力下降,可能通过TGF-β/smad3信号通路逆转EMT有关。

Objective To observe the effect of ORAI1 on cell migration and invasion in prostate cancer PC-3 cells,and explore whether ORAI1 contribute to reverse the epithelial-mesenchymal transition(EMT)transformation.Methods PC-3 cells were transfected with lentiviral vector LV-ORAI1-RNAi(KD group)and related negative vector(NC group),the transforming growth factor-β1(TGF-β1)was added separately.After transfection,the expression of ORAI1 in PC-3 cells was detected by real-time reverse transcriptase-polymerase chain reaction(RT-qPCR)and Western blot.The cell mobility,migration and invasion ability of PC-3 cells were measured by Celigo scratch assay,transwell migration and invasion assays,respectively.The expression of EMT markers[E-cadherin,N-cadherin,Vimentin],and smad3,p-smad3 were detected by Western blot analysis.Results After transfected with LV-ORAI1-RNAi,ORAI1 expression in PC-3 cells was decreased at both mRNA and protein levels(P<0.05).Celigo scratch assay showed that the mobility of KD group was(10.24%±1.17%)decreased(P<0.05)compared with that of NC group(14.38%±0.77%),the mobility of KD+TGF-β1 group(12.44%±1.29%)was decreased(P<0.05)compared with that of NC+TGF-β1 group(26.21%±2.23%).Transwell migration assay showed that the number of migrating cells in KD group(229+8.4)was lower than that in NC group(298+14.2)(P<0.05);compared with NC+TGF-β1 group(338+12.9),the number of migrating cells in KD+TGF-β1 group(216+12.2)was lower(P<0.05).Transwell invasion assay showed that the number of invasive cells in KD group(46+3.4)was lower than that in NC group(79+4.6)(P<0.05);compared with NC+TGF-β1 group(123+5.1),the number of invasive cells in KD+TGF-β1 group(66+5.5)was lower(P<0.05).Western blot results showed that the expression of E-cadherin decreased,while the expression of N-cadherin and Vimentin increased in NC+TGF-β1 group,compared with NC group.Compared with NC+TGF-β1 group,the expression of E-cadherin increased,while the expression of N-cadherin,Vimentin and p-smad3 decreased in KD+TGF-β1 group.Conclusions The decreased expression of ORAI1 in PC-3 cells may be related to the reversal of EMT through TGF-β/smad3 signaling pathway.