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目的:研究siRNA(small interference RNA)对人膀胱癌BIU-87细胞凋亡和端粒酶催化亚单位(hTERT)基因表达的影响。方法:设计并体外转录合成针对hTERT基因的3个特异性siRNA,通过脂质体将siRNA转入BIU-87细胞,分别采用MTT和DNA原位末端标记(TUNEL)法检测siRNA对BIU-87细胞生长抑制率(IR%)和凋亡指数(AI%)的影响,半定量逆转录聚合酶链反应(RT-PCR)和Western Blotting检测siRNA对hTERT mRNA及其蛋白表达的影响。结果:转染后的siRNA1～3组的BIU-87细胞的IR(34.62%、62.19%、57.43%)和AI(30.62%、54.26%、51.50%)均分别显著高于正常对照组(3.46%和5.03%)(均P<0.05),hTERT mRNA及其蛋白表达水平均显著低于对照组;其中siRNA 2～3对BIU-87细胞的IR、AI和hTERT表达的抑制作用均显著高于siRNAl。结论:体外转录合成的siRNA可抑制BIU-87细胞hTERT的表达,诱导BIU-87细胞凋亡,从而抑制BIU-87细胞生长,为siRNA介导的膀胱肿瘤基因沉默提供实验依据。
AIM: To investigate the effect of siRNA on apoptosis and expression of telomerase catalytic subunit (hTERT) genes in human bladder cancer cell line BIU-87. Methods: Three siRNA specific for hTERT gene were designed and synthesized in vitro. The siRNA was transfected into BIU-87 cells by lipofectamine. The expression of Bcl-2 mRNA in BIU-87 cells was detected by MTT assay and TUNEL assay. (IR%) and apoptotic index (AI%). The effects of siRNA on hTERT mRNA and protein expression were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western Blotting. Results: After transfection, the percentage of IR (34.62%, 62.19%, 57.43%) and AI (30.62%, 54.26%, 51.50%) in BIU-87 cells of siRNA1-3 group were significantly higher than that of the normal control group And 5.03%, respectively) (both P <0.05). The mRNA and protein expression levels of hTERT in both BIU-87 cells and siRNA groups were significantly lower than those in siRNA group. SiRNA 2 and 3 inhibited the expression of IR, AI and hTERT significantly . CONCLUSION: siRNA synthesized in vitro can inhibit the expression of hTERT in BIU-87 cells and induce the apoptosis of BIU-87 cells, thereby inhibiting the growth of BIU-87 cells and providing an experimental basis for siRNA-mediated bladder tumor gene silencing.