CHK1 shRNA对HeLa细胞凋亡和细胞周期的影响

The Role of CHK1 Gene Silence in Apoptosis and Cell Cycle of HeLa Cells

目的采用RNA干扰(RNAinterference,RNAi)技术沉默CHK1基因的表达,观察其对高表达CHK1的宫颈癌细胞系HeLa细胞增殖、凋亡的影响。方法构建针对CHK1的短发夹状RNA(short hairpin RNA,shRNA)真核表达质粒,将该质粒通过脂质体转染法导入HeLa细胞,以RT-PCR和Western blot法分别检测其CHK1基因和蛋白的表达,流式细胞仪检测细胞凋亡和细胞周期的变化,MTT法检测细胞的增殖。结果shRNA能明显降低HeLa细胞表面CHK1的表达,与对照组和空载体转染组相比,shRNA转染组HeLa细胞CHK1 mRNA水平下降了66%,蛋白水平下降了60%(P<0.05)。此外,shRNA转染组的HeLa细胞凋亡率明显增加,增殖活性明显降低,而且G2/M期细胞百分率显著下降。对照组、空载体转染组和shRNA转染组的G2/M期细胞百分率分别为(53.96±1.35)%、(54.08±1.39)%和(15.10±0.87)%(P<0.05)。结论CHK1shRNA能明显增加HeLa细胞的凋亡,抑制该细胞的增殖,削弱其G2/M期阻滞,为进一步研究CHK1在肿瘤治疗中的作用机制提供实验基础。

Objective To investigate the effect of CHK1 gene silence on apoptosis, cells viability and cell cycle of cervix carcinoma HeLa cells highly expressing CHK1. Methods Short hairpin RNA targeting at checkpoint kinasel （CHK1） gene was constructed and transfected into HeLa cells. The CHK1 expression in HeLa cells was detected by using RT-PCR and Western blot. Apoptosis and cell cycle of HeLa cells were assayed by flow cytometry. Cell viability was tested by MTT assay. Results The expression levels of CHK1 mRNA and protein in CHK1 shRNA-transfected group were reduced by about 66% and 60 % respectively, as compared with those in control group and empty vector-transfected group （P〈0.05）. Compared with control group and empty vector-transfected group, apoptosis rate of HeLa cells transfected with shRNA was remarkably increased, while cell viability in those cells was decreased significantly （P〈0.05）. In addition, the percentage of the cells in G2/ M phase was （15.10±0.87）%, which was lower than that in the remaining two groups [-（53.96± 1.35）% and （54.08± 1.39） % respectively, P〈0.05]. Conclusion shRNA could obviously decrease the CHK1 expression in HeLa cells, increase their apoptosis, decrease their viability and weaken the G2/M phase block of cell cycle.