沉默survivin基因表达对人宫颈癌hela细胞恶性行为的影响

Effect of specific inhibition of survivin gene expression by small interfering RNA on the malignant behavior of human cervical cancer cell line Hela

目的研究小干扰RNA(small interferingRNA,siRNA)抑制survivin基因的表达对人宫颈癌Hela细胞凋亡的影响。方法根据survivin基因设计序列特异性的siRNA(survivin-siRNA),在脂质体介导下转染Hela细胞,Westernblot方法检测survivin蛋白表达情况,利用TUNEL实验、Annexin-V实验和流式细胞仪方法检测细胞转染前后凋亡的形态学的变化、凋亡率及凋亡周期的变化,Western blot检测caspase-3、caspase-7在转染前后细胞中表达的变化。结果转染survivin-siRNA后,Hela细胞survivin蛋白表达明显下降,与空白对照组及阴性对照组相比,能明显诱导细胞凋亡,细胞被阻滞在G/M期(p<0.05)。转染后Hela细胞中caspase-3、caspase-7蛋2白表达比空白对照组及阴性对照组明显增强(p<0.05)。结论 Survivin-siRNA转染人宫颈癌Hela细胞可上调细胞中survivin蛋白的表达水平,诱导细胞凋亡,与caspase-3、caspase-7的上调有关。

Objective To study the effect of specific inhibition of survivin gene expression by small interfering RNA（siRNA） on the apoptosis of human cervical cancer cell line Hela. Methods The survivin siRNA was designed by web based software, synthesized and then transfected into hela cell line by lipofectamine 2000 reagent. The survivin gene expression inhibition and the expression of caspase-3 and caspase-7 proteins was assessed by Western blot, the apoptosis rate and cell cycle were assessed by TUNEL, Annexin-V and flow cytometry. Results Significant downregulation of the survivin protein expression level was found after transfection of the survivin-siRNA in Hela cells. The apoptosis rate of Hela cells was notably increased, Hela ceils were arrested in G2/M phase, the protein levels of caspase-3 and caspase-7 proteins were higher in siRNA transfected hela cells compared with the untransfected cells and negative control transfected cells. Conclusion Transient transfection of synthesized survivin-siRNA in vitro can downregulate the expression of survivin protein in Hela ceils and induce the apoptosis of hela cells, which may be related to the upregulation of caspase-3 and caspase-7 expressions.