慢病毒介导的RNA干扰沉默Slug基因对结肠癌HCT116细胞增殖和凋亡的影响

Effects of Slug RNA interference by lentivirus on proliferation and apoptosis of colon carcinoma cells HCT116

目的:探讨利用RNA干扰(RNA interference,RNAi)技术沉默Slug基因,观察对结肠癌HCT116细胞增殖和周期的影响。方法:构建Slug基因特异性siRNA慢病毒载体,感染结肠癌HCT116细胞,设立空白对照组、阴性对照组及SlugsiRNA三组,应用Real-time PCR和Western blot方法分别从基因和蛋白质水平检测各组干扰质粒对Slug基因的干扰效果,MTT法检测Slug基因在siRNA作用下的细胞增殖率,流式细胞仪检测细胞凋亡周期变化情况。结果:转染Slug siRNA后,结肠癌HCT116细胞中Slug基因mRNA和蛋白表达明显受到抑制(P<0.05);MTT检测,干扰组细胞增殖水平明显低于阴性对照组;流式细胞仪检测细胞G1期细胞百分比(52.3±0.6)高于阴性对照组(45.1±0.3,P<0.05)。结论:Slug siRNA能明显下调靶基因Slug的表达,在体外可抑制结肠癌HCT116细胞的生长并促进其凋亡。

Objective： To investigate the inhibitory effect of Slug gene short interfering RNA （siRNA） on proliferation and cell cycle of human colon carcinoma HCT116 cell line. nethods：SiRNA targeting Slug gene was constructed into lentivirus vector, and siRNA targeting Slug was transfected into colon cancer cell line HCT116 in vitro. Non interference group, negative siRNA group and RNA interference group were set up. Western blot was used to detect the expression of Slug protein in the transfected cells, and to detection of Slug mRNA expression by Real-time PCR. Cell proliferation was measured by MTF test. Cell cycle was detected by flow cytometry with Annexin V/PI. Results ： Slug siRNA significantly inhibited the expression of Slug in colon cancer ceUs at both mRNA and protein levels. The apoptotic rate of HCT116 cells transfected with siRNA was significantly higher than that of the control cells （ P 〈 0.05 ）. Compared with negative control group ,the proliferation rate of HCT116 cells was decreased by MTr assay. The percentage of cells at G1 phase（52.3 ± 0.6） in RNAi group was increased compared to negative control group（45.1 ± 0.3） （P 〈 0.05 ）. Conclusion：Direct inhibition of Slug expression by siRNA significantly suppresses the proliferation and promotes apoptosis of HCT116 cells in vitro, indicating a new approach of gene therapy for colon carcinoma.