shRNA介导的波形蛋白基因沉默对肝癌细胞迁移、侵袭能力的影响
Effect of shRNA lentiviral mediated Vimentin gene silencing on migration and invasion of hepatocellular carcinoma cells

目的:探讨shRNA介导的波形蛋白(Vimentin)基因沉默对肝癌细胞迁移、侵袭能力的影响。方法:将体外培养的肝癌Huh7细胞分为对照组(未感染)、阴性对照组(感染慢病毒载体LV-NC-GFP-shRNA)和实验组(感染慢病毒LV-Vimentin-GFP-shRNA),采用RT-PCR检测3组细胞中Vimentin mRNA的表达,Transwell小室实验检测细胞的迁移和侵袭能力,Western blot法检测细胞中Vimentin、MMP-9和受体酪氨酸激酶(AXL)蛋白的表达。结果:成功构建了慢病毒LV-Vimentin-GFP-shRNA,建立了Vimentin基因稳定沉默的Huh7细胞。Vimentin基因稳定沉默的实验组细胞中Vimentin mRNA和蛋白的表达均降低,迁移和侵袭细胞数减少,MMP-9和AXL蛋白的表达减弱,与对照组和阴性对照组相比,差异均有统计学意义(P<0.05)。结论: Vimentin基因沉默能够抑制肝癌Huh7细胞的迁移和侵袭。
Aim:To investigate the effect of shRNA lentiviral mediated Vimentin gene silencing on migration and invasion of hepatocellular carcinoma cells.Methods:Hepatoma Huh7 cells cultured in vitro were divided into control group(uninfected), negative control group(infected with LV-NC-GFP-shRNA)and experimental group(infected with LV-Vimentin-GFP-shRNA).The expression of Vimentin mRNA in Huh7 cells was detected by RT-PCR.The migration and invasion of Huh7 cells were measured by Transwell cell experiment.The expressions of Vimentin,MMP-9 and receptor tyrosine kinase(AXL)proteins in Huh7 cells were tested by Western blot method.Results:The lentiviral vector targeting Vimentin gene LV-Vimentin-GFP-shRNA was successfully constructed,and Vimentin gene stable silencing Huh7 cells were established.Compared with the control group and the negative control group,the expression of Vimentin mRNA and protein reduced significantly,the number of cell migration and invasion cut down,and the expressions of MMP-9 and AXL proteins decreased in the experimental group(P<0.05).Conclusion:Vimentin gene silencing could inhibit the migration and invasion of liver cancer Huh7 cells