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目的探讨乙肝病毒X基因(HBX)在肝细胞癌(HCC)侵袭转移中的作用。方法利用psiRNA-hH1neo质粒,构建针对HBX的shRNA表达载体psiRNA1、psiRNA2、psiRNA3,转染肝癌细胞株HepG2.215,用荧光定量PCR仪检测siRNA对HBX mRNA表达的抑制作用,用Western blot法检测siRNA对HBX蛋白表达的抑制作用。以Transwell小室测定HepG2.215细胞与纤维连接蛋白(Fn)的体外黏附能力。结果成功构建shRNA表达载体,shRNA表达载体均可不同程度地抑制HBX的表达,其中psiRNA1对HBX的抑制作用最强,对HBX mRNA抑制率达80.27%,对HBX蛋白的抑制率为65.59%;细胞体外黏附能力受到明显抑制,在培养至48、72h后,对照组和转染组跨膜细胞数分别为442.6±57.1vs376.4±55.2和588.2±70.1vs513.6±68.5,两时相点均有明显差异(P<0.05)。结论阻断HBX的表达可明显抑制肝癌细胞HepG2.215的体外侵袭性生长。
Objective To investigate the role of hepatitis B virus (HBV) gene in the invasion and metastasis of hepatocellular carcinoma (HCC). Methods psiRNA-hH1neo plasmid was used to construct siRNA expression vectors psiRNA1, psiRNA2 and psiRNA3 targeting HBX and transfected into HepG2.215 cells. The inhibitory effect of siRNA on HBX mRNA expression was detected by fluorescence quantitative PCR, and siRNA was detected by Western blot Inhibition of HBX protein expression. The in vitro adhesion of HepG2.215 cells to fibronectin (Fn) was measured in a Transwell chamber. Results The shRNA expression vector was successfully constructed. ShRNA expression vector could inhibit the expression of HBX to some extent. Among them, psiRNA1 had the strongest inhibitory effect on HBX, the inhibitory rate of HBX mRNA was 80.27% and the inhibitory rate of HBX protein was 65.59% The adhesion capacity in vitro was significantly inhibited. At 48 and 72 hours after culture, the number of transmembrane cells in the control group and transfection group were 442.6 ± 57.1 vs 37.4 ± 55.2 and 588.2 ± 70.1 vs 513.6 ± 68.5, respectively, There was a significant difference (P <0.05). Conclusion Blocking the expression of HBX can significantly inhibit the invasive growth of HepG2.215 cells in vitro.