siRNA干扰ski基因的表达对人肝癌HepG2细胞生物学功能的影响

Effect of ski-siRNA on biological function of human hepatoma cell line HepG2

目的:设计并化学合成针对原癌基因ski的siRNA分子片段,转染人肝癌HepG2细胞,观察其对HepG2细胞增殖、细胞周期和凋亡等生物学功能方面的影响。方法:设计并化学合成针对原癌基因ski的3个siRNA分子序列,阳离子脂质体法瞬间转染HepG2细胞,运用实时定量PCR法和Western印迹法检测细胞中ski基因在mRNA水平和蛋白水平的变化。然后利用MTT法和流式细胞术检测转染ski-siRNA的HepG2细胞增殖、细胞周期和凋亡等生物学功能方面指标的变化。结果:3对特异性ski-siRNA均有效地抑制了ski基因的表达,以siRNA-B抑制效果最好,可达到70%,而且随着转染时间的延长,ski的表达呈逐步下降趋势。转染ski-siRNA后HepG2细胞的增殖能力明显受到抑制(P<0.05),S期细胞明显减少,是阴性对照组的2倍以上。结论:靶向ski基因的siRNA分子片段可以有效地抑制人肝癌HepG2细胞的生长,使进入S期的细胞明显减少,其作用与下调ski基因的表达有关,ski可能是肝癌治疗的一个潜在靶点。

Objective： To design and prepare siRNAs targeting ski gene and to observe its influence on the biological functions of HepG2 cells, such as proliferation,cell cycle, apoptosis, etc.. Methods： Three specific siRNAs of ski were designed and synthesized,and were transiently transfected into HepG2 via cathodolyte liposome transfection method. Real-time PCR and Western blotting were used to measure ski expression at mRNA and protein levels. The cell proliferation was assessed by MTT assay and the changes in cell cycle and apoptosis were evaluated by flow cytometry. Results： All the 3 specific ski-siRNA（A,B, C） effectively inhibited the expression of ski gene, with ski-siRNA-B having the highest inhibition rate（70 % ）. Furthermore, the ski expression had a decreasing tendency with the transfection time. The proliferation of HepG2 cells was markedly inhibited by ski-siRNAs（P〈0.05）; the number of cells at S stage was obviously decreased,being 2 folds that of the negative control group. Conclusion： The siRNA of ski gene can effectively induce growth inhibition of HepG2 cells and reduce cells of S stage, which is possibly through down-regulation of ski gene.