人类P100蛋白表达抑制稳定株的建立及其对HeLa细胞周期的影响

Effects of the Stable Down-Expression of Human P100 Protein on Cell Cycle in HeLa Cell Line

目的构建针对人类P100(hP100)基因的小干扰RNA(small interfering RNA,siRNA)质粒,转染后筛选P100表达抑制的稳定细胞株并鉴定其表达情况;同时检测其对细胞周期的影响。方法设计针对hP100基因的siRNA序列,构建pGenesil-shRNA-hP100质粒(smallhairpinRNA,shRNA,小发夹RNA),经测序鉴定后转染入宫颈癌细胞HeLa;经G418筛选出稳定株;Western印迹检测稳定株之抑制率。流式细胞术检测hP100蛋白表达抑制对细胞周期的影响。结果成功构建了针对hP100的siRNA质粒,并用之筛选出hP100蛋白表达抑制的稳定株,瞬时转染及稳定株hP100表达明显降低。流式细胞术显示抑制hP100蛋白表达使G1期细胞比例增加,S期和G2/M期细胞比例降低。结论RNAi(RNA interference,RNA干扰)可持续稳定地抑制人类P100蛋白的表达,人类P100蛋白表达抑制对细胞周期有调节作用。

Objective To suppress the expression of human P100 gene by RNA interference, screen the stable transfectant and observe its influence on cell cycle. Methods The stable screening inhibition of RNAi was used to stably suppress the expression of P100 protein. After small hairpin RNA （shRNA） targeting human P100 gene was designed, recombinant vector pGenesil-shRNA- hP100 was constructed and transfected into Hela cells, which were selected by using C,418 to establish stable transfectant, hP100 expression was detected by Western blotting and the suppression efficiency was calculated in transiently transfected cells and stable transfectant separately. The cell cycle distribution was detected by flow cytometry （FCM） . Results pGenesil-shRNA-hP100 plasmid was constructed successfully, and the hP100 expression in both transient transfection and stable transfectant were obviously suppressed as compared with that in negative control group. Compared with the control, cell cycle of the stable transfectant was prolonged in G1 phase. Conclusion RNAi may continually and stably suppress hP100 protein expression and the inhibited expression of P100 protein could regulate cell cycle in HeLa cell line.