RNA干扰技术对HeLa细胞hTERT基因的抑制作用

目的 探讨RNA干扰对宫颈癌HeLa细胞人端粒酶反转录酶基因的抑制效应。方法 化学合成靶向于hTERT基因的siRNA,用阳离子脂质体转染法将其导入宫颈癌细胞内。实验分组:转染组siRNA-1、siRNA-2、siRNA-3、siRNA-4、阴性对照组(siRNA-NC)、空白对照组(Blank)。通过RT-PCR和Western Blot方法分别检测宫颈癌细胞转染后细胞hTERT mRNA和蛋白水平,MTS法检测细胞生长增殖,流式细胞仪检测细胞凋亡情况。结果 siRNA-3组的端粒酶活性明显下降,hTERT mRNA表达水平显著下降,hTERT蛋白表达明显下降,细胞凋亡率明显升高,与其他各组比较,差异有统计学意义(P＜0.01)。结论 运用RNAi干扰技术,以hTERT基因为靶点的siRNA能特异性抑制宫颈癌HeLa细胞hTERT基因,下调hTERT mRNA及蛋白表达水平,抑制宫颈癌细胞生长增殖,促进宫颈癌细胞凋亡,为宫颈癌的基因治疗研究提供实验依据。
Objective To investigate the effect of RNA interference(RNAi) targeting human telomerase reverse transcriptase(hTERT) gene on cell apoptosis in cervical carcinoma HeLa cells. Methods Short interfering RNAs(siRNAs) targeting hTERT gene were synthesized, and siRNAs were transfected into Hela cells by cationic lipofection. Cervical carcinoma Hela cells were divided into six groups:blank control group, siRNA-NC group(negative group), siRNA-1, siRNA-2, siRNA-3, and siRNA-4 groups(RNA interference groups). The telomerase hTERT mRNA and hTERT protein were detected by reverse transcriptase polymerase chain reaction(RT-PCR) and Western blot, respectively. The proliferation of the Hela cell was analyzed by MTS and cell apoptosis was examined by flow cytometry. Results The hTERT mRNA of siRNA-1, siRNA-2, siRNA-3, and siRNA-4 groups, siRNA-NC group and blank group were 1.779±0.42,1.408±0.16,0.463±0.14,0.986±0.08,0.987±0.08,1.717±0.08, respectively. Compared to the other groups, the relative expression level of the hTERT mRNA of siRNA-3 group was declined markedly(P＜0.01). The relative activities of the hTERT protein of siRNA-1, siRNA-2, siRNA-3, and siRNA-4 groups, siRNA-NC group and blank group were(85.64±3.28)%,(70.67±3.40)%,(38.64±5.28)%,(88.87±3.39)%,(89.64±4.18)%,(99.67±5.40)%, respectively. In comparison with other groups, the expression level of hTERT protein in siRNA-3 group was declined significantly (P＜0.01). The apoptosis rate of siRNA-3, siRNA-NC group and blank group were(48.21±4.25)%,(8.67±0.40)%,(8.64±0.28)%, respectively. Compared with other groups, the apoptosis rate of siRNA-3 group was elevated significantly(P＜0.01). Conclusion RNAi can specifically silence the hTERT gene of cervical carcinoma Hela cells by down-regulating the expression of hTERT