RNAi沉默HSP70基因对宫颈癌HeLa细胞的影响

The effect of RNAi silencing HSP70 gene on cervical cancer HeLa cells

目的:本研究初步探讨RNAi技术应用于宫颈癌基因治疗的特异性及其作用机理。方法:设计靶向HSP70基因siRNA序列,构建重组表达载体pTZU6+1-siRNA-HSP70,在脂质体介导下瞬时转染宫颈癌HeLa细胞,在体外诱导RNAi沉默HSP70基因,同时设转染空载体pTZU6+1为阴性对照组和不加任何试剂的空白对照组。采用RT-PCR和Western blotting方法检测实验组和对照组中HSP70表达;MTT法检测细胞生长与增殖;流式细胞术检测细胞的凋亡率和细胞周期分布。结果:与对照组相比,实验组转染HeLa细胞后,细胞的HSP70 mRNA和蛋白表达均明显下调(P<0.01);细胞生长抑制率增高(P<0.05);细胞周期相分布发生变化。结论:siRNA明显抑制HSP70的表达及宫颈癌HeLa细胞的增殖,并引起细胞凋亡和细胞周期改变,为进一步研究HSP70基因在宫颈癌细胞凋亡中的作用及其机制奠定了基础。

Objective：To explore the application of RNAi technology in gene therapy of cervical cancer and its mechanism.Methods： Small interference RNA targeting HSP70 gene was designed,and was cloned into the vector pTZU6 ＋1 to construct pTZU6 ＋1-siRNA-HSP70.Then pTZU6 ＋1-siRNA-HSP70 was transfected into HeLa cells through lipofectamineTM 2000,at the same time,pTZU6 ＋1 was transfected as the negative control and that without any reagent as the blank control.The expression levels of HSP70 mRNA and protein were detected respectively by semi-quantity RT-PCR and Western blotting.The inhibition ratio of HeLa cells was determined by MTT.The apoptosis rate and cell cycle was detected by flow cytometry.Results： Compared with the control group,the expression levels of HSP70 mRNA and protein in HeLa cells were significantly decreased（P〈0.01）,cell growth inhibition rate was significantly higher（P〈0.05）,cell cycle phase distribution changed.Conclusion： siRNA can inhibit the expression of HSP70 and HeLa cells proliferation,promote HeLa cell apoptosis and change cell cycle phase,which lays foundation for further study of HSP70 gene in cervical cancer cell apoptosis and its mechanism.