小干扰RNA沉默组织因子基因对肝癌侵袭转移能力的影响

目的 应用RNA干扰(RNAi)技术抑制人类肝癌细胞内组织因子(TF)基因表达,从而探讨对其侵袭转移能力的影响。方法 本实验以人类肝癌细胞系HepG2细胞株为实验对象,将携带有针对TF小干扰RNA( siRNA)序列的质粒pGPU6/GFP/Neo-TF siRNA,转染HepG2细胞株,以适当浓度的G418筛选获得阳性克隆,通过RT-PCR和蛋白质印迹法比较转染TF siRNA(+)质粒、转染TF siRNA( -)质粒以及未转染任何质粒的HepG2细胞内源性TF在基因及蛋白质表达水平的差异,进而应用Transwell体外侵袭实验检测三者的侵袭转移能力,探讨TF对肿瘤细胞侵袭转移能力的影响。结果 (1) RT-PCR结果 显示,转染TF siRNA(+)质粒的HepG2细胞其内源性TF表达水平低于转染TF siRNA（-）质粒和未转染质粒的表达水平。(2)蛋白质Western印迹法结果 显示,转染TFsiRNA(+)质粒的HepG2细胞其内源性TF蛋白表达水平低于转染TF siRNA( -)质粒和未转染质粒的表达水平。(3) Transwell体外侵袭实验结果 显示,转染TF siRNA(+)质粒的HepG2穿膜细胞数(14±10)个]少于转染TF siRNA（-）质粒的HepG2穿膜细胞数(128±18)个],经t检验比较,两组差异有统计学意义（P＜0.05）。这提示应用RNA干扰技术抑制肿瘤细胞TF表达可以使HepG2细胞侵袭转移能力显著下降。结论 TF与肝癌细胞的侵袭转移能力密切相关,应用RNA干扰技术抑制其表达可以明显降低细胞HepG2体外侵袭转移能力。

Effect of siRNA silenced human tissue factor gene on hepatocellular carcinoma cell invasion and metastasis

Objective To study the effect of tissue factor (TF) on human hepatocellular carcinoma cells when their inner TF gene was knocked down by RNA interference. Methods The eukaryotic expression vector bearing siRNA sequence that targeted at TF gene was transfected into human hepatocellular carcinoma cell line HepG2. RT-PCR and Western blot were used to detect the changes of TF gene expression. Transwell invasion assay invitro were used to show the invasive ability of HepG2 cells with transfected pGPU6/GFP/Neo-TFsiRNA. Results RT-PCR and Western blot analysis showed that HepG2 cells with pGPU6/GFP/Neo-TFsiRNA transfected decreased the endogenous TF gene expression. Correspondingly the mean invading cells was 14 ± 10 for pGPU6/GFP/Neo-TFsiRNA transfection and 128 ± 18 for the NCsiRNA transfection,respectively by the Transwell invasion test in vitro( P ＜0.05 ). The results indicated that the invasive ability of the HepG2 cells with pGPU6/GFP/Neo-TFsiRNA transfected decreased obviously. Conclusions TF is involved in the progression of hepatocellular carcinoma and inhibiting the expression of TF can decrease the invasion and metastasis ability of tumor cell in vitro.