甲磺酸盐及紫外线敏感性81号基因沉默对人结肠癌HCT116细胞增殖凋亡的影响

Effects of methyl methansulfonate, ultraviolet sensitive 81 gene silencing on proliferation and apoptosis of human colon cancer HCT116 cells

目的 观察甲磺酸盐及紫外线敏感性81号基因（Mus81）沉默对结肠癌HCT116细胞增殖和凋亡的影响并探讨其调控机制.方法 采用慢病毒介导的小干扰RNA （siRNA）技术靶向沉默Mus81基因的表达,以实时定量聚合酶链反应（Real-time PCR）及Western blot检测Mus81基因的表达抑制率.采用噻唑蓝（MTT）法绘制细胞生长曲线,以碘化丙锭（PI）及膜联蛋白V（Annexin V）-APC染色法检测细胞周期及凋亡率,以Real-time PCR法检测B细胞淋巴瘤/白血病-2（bcl-2）、bcl-2相关X蛋白（bax）及p53基因的表达.结果 Mus81基因和蛋白的抑制率分别为84.64％和71.03％ （P＜0.01）.Mus81沉默后HCT116细胞的增殖速度明显降低（P＜0.01）,凋亡率明显升高[（19.69±0.20）％比（8.74±0.11）％,P＜0.01],G2/M期细胞比例轻微上升[（12.99±0.48）％比（11.69±0.41）％,P＜0.05],其bax和p53的表达分别升高了71.73％和12.02％ （P ＜0.05）,bcl-2表达则降低了37.72％ （P ＜0.01）.结论 Mus81基因沉默可明显促进HCT116细胞凋亡并抑制其增殖,其机制可能与调控bax、bcl-2及p53基因的表达有关.

Objective To investigate the effects of methyl methansulfonate,ultraviolet sensitive 81 gene （Mus81 gene） silencing on proliferation and apoptosis of human colon cancer HCT116 cells.Methods Lentivirus-mediated small interfering RNA （siRNA） was employed to inhibit Mus81 expression in HCT116 cells.Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting were used to determine the inhibition efficiency of Mus81 expression.Growth curves of HCT116 cells were established by methyl thiazol tetrazolium （MTT） assay.Cell cycle distribution and apoptosis were detected by propidium iodide （PI） and Annexin V-APC staining.Expression levels of B cell lymphoma/leukemia-2 （bcl-2）,bcl-2 associated X protein （bax） and p53 genes were also determined by real-time quantitative polymerase chain reaction （Real-time PCR）.Results The interfering efficiency of Mus81 gene and protein was 84.64% and 71.03% respectively （P 〈0.01）.Mter Mus81 gene silencing,the growth rate of HCT116 cells was decreased significantly （P 〈 0.01）,but the apoptosis rate increased significantly [（19.69 ± 0.20） % vs.（8.74 ± 0.11） %,P 〈 0.01].The proportion of Mus81 depleted HCT116 cells at G2/M phase was slightly increased [（12.99 ± 0.48） % vs.（11.69 ± 0.41） %,P 〈 0.05].The expression of bax and p53 was increased significantly by 71.73% and 12.02% in Mus81 silenced HCT116 cells but bcl-2 expression was decreased significantly by 37.72% （P 〈 0.01）.Conclusion Mus81 silencing inhibits the proliferation of human colon cancer HCT116 cells by promoting cell apoptosis through regulating the expression of bax,bcl-2 and p53.