Bmi-1 siRNA对宫颈癌Hela细胞体外增殖能力的影响

Effects of siRNA silencing of Bmi-1 expression on proliferation of Hela cells

关键词:  Bmi-1基因  沉默  Hela细胞  增殖

Key words:  Bmi-1 gene  silencing  Hela cells  proliferation

摘要:

[目的]观察siRNA沉默Bmi-1表达对Hela细胞增殖能力的影响。[方法] 构建了表达Bmi-1 siRNA的重组真核表达载体，将其转染入Hela细胞，利用荧光法观察转染效率。用RT-PCR及Western blot方法检测转染后细胞Bmi-1 mRNA及Bmi-1蛋白的表达情况；用MTT比色法、台盼蓝拒染法检测Bmi-1 siRNA对Hela细胞增殖的抑制作用；用流式细胞仪分析各组细胞的细胞周期；用平板克隆形成实验检测Bmi-1 siRNA对Hela细胞的单细胞增殖能力的影响；应用免疫细胞化学（SP法）及Western blot法检测各组细胞Ki-67及CyclinD1的表达。[结果]将构建好的重组质粒成功转染进入了Hela细胞中且转染效率在90%左右；Bmi-1 siRNA转染Hela细胞Bmi-1 mRNA及Bmi-1蛋白表达沉默，Bmi-1表达沉默后抑制Hela细胞增殖并使细胞周期阻滞于G0-G1期，能明显抑制Hela细胞的单细胞增殖能力；同时Ki-67及CyclinD1的表达均明显下降。[结论] siRNA介导的Bmi-1基因的表达沉默能抑制Hela细胞的增殖能力，Bmi-1表达可能与宫颈癌的发生发展相关。

Abstract:

[Objective]The effects of silencing the oncogene Bmi-1 on proliferative capacity of Hela cells with siRNAs targeted to Bmi-1 were explored.[Methods]The Bmi-1 siRNA expression vectors were constructed and transfected into the Hela cells with lipofectamine method. Neo gene was detected by RT-PCR for confirming the success of transfection, and the efficiency of transfection was observed by measuring expression of EGFP in Hela cells.The levels of Bmi-1 mRNA and its protein in Hela cells transfected with Bmi-1 siRNA were analyzed by RT-PCR and Western blot respectively. The proliferation of Hela cells transfected with Bmi-1 siRNA was analyzed with MTT and Typan blue exclusion methods. Flow cytometry was used for detecting cell cycle. Plate colony forming assay was used to analyze single-cell proliferative capacity. Ki-67 and CyclinD1 were examined with immunooytochemistry stain and Western blotting respectively. Finally, the abilitie of tumorigenesis of Hela cells transfected with Bmi-1 siRNA was observed in nude mouse respectively. [Results]The recombinant plasmids could be successfully transfected to and expressed in HeLa cells, the efficiency of transfection was estimated to be about 90%. The Bmi-1 siRNAs were proved to silence mRNA and protein expressions in tranfected Hela cells without inducing cells death in vitro. The transfected Hela cells were arrested in G0-G1 phase. The single-cell proliferative capacity of Bmi-1 siRNA transfected Hela cells was inhibited. The expressions of Ki-67 and CyclinD1in the transfected Hela cells were significantly attenuated compared to those of the control groups.[Conclusion]Silencing Bmi-1 gene through siRNA inhibits the Hela cells are to moliferate. The expression of Bmi-1 is associated with carcinogenesis in cervical carcinoma.