siRNA端粒酶催化亚单位诱导人膀胱癌BIU-87细胞凋亡的研究

Study of siRNA HTERT Gene Inducing the Cell Apoptosis of Human Bladder Cancer Cell Line BIU-87

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。

Objective：To study the influence of siRNA （small interference RNA） on the cell apoptosis in human bladder cancer cell line BIU-87 and bTERT gene expression. Methods：3 specific siRNA targeted hTERT gene were synthesized after designed and transcription in vitro. After siRNA were transferred into BIU-87 cells by liposome, MTT and TUNEL methods were used to detect the proliferation inhibitory rate （IR） % and apoptosis index （AI） % of BIU-87 cells, semi-quantitive reverse transcription polymerase chain reaction （RT-PCR） and Western Blotting techniques were used to examine the effects of siRNA on the expressions of the hTERT gene and protein. Results.. The IR （34.62%, 62.19%0 and 57.43%）and AI （30.62%, 54.26% and 51.50%） of BIU-87 cell in the siRNA 1-3 group were respectively significantly higher than those （3.46% and 5.03% ） in the control （P〈0.05）, and the expressions of hTERT mRNA and protein in the siRNA groups were all lower than that in the control. Moreover, the effects of siRNA2 and siRNA 3 on inhibiting hTERT expression, IR and AI of BIU-87 cells were stronger than the group of siRNA1. Conclusions： The siRNA of transcription in vitro can significantly inhibit the expression of hTERT, induce cell apoptosis and inhibit the growth of BIU-87 cells which may provide the experimental evidence for the mechanism of the bladder tumor gene silencing therapy by siRNA.