乙肝病毒X基因阻断降低肝癌细胞HepG2.215与纤维连接蛋白的黏附

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。

HBX Expression Inhibtion by RNA Interference Suppress on Invasive Growth of Human Hepatocellular Carcinoma Cells HepG2.215 in vitro

Objective To study inhibitory effects of Hepatitis B virus X gene（ HBX） expression inhibition on the adhesion of HepG2.215 hepatocellular carcinoma cells（HCC） with fibronectin（Fn） in vitro. Methods Based on the plasmid psiRNA-hH1neo,three plasmid-derived siRNAs which complemented to the coding region of the HBX gene were prepared,which were named psiRNA1、psiRNA2、 psiRNA3 respectively. The above siRNAs were transfected into HepG2.215 cells （clonal cells derived from HepG2 cells that contain integrated HBV ayw DNA）.The HBX mRNA quantities were measured by quantitative real-time PCR,and the level of HBX protein was quantified by using Western blot analysis. The invasive activity of tumor cells was assayed in a Transwell cell culture chamber. Results The results demonstrated that plasmid-derived siRNAs could effectively reduce the quantities of HBX mRNA and protein. The plasmid-derived siRNA psiRNA1 was found to be the most effective inhibitor of HBX expression. It could inhibit the expression of HBX protein by 65.59% and suppress HBX mRNA by 80.27% . A lower rate of invasion in vitro detected in the transwell cell culture chamber was also observed in the group treated with transfection of psiRNA1,and the number of cells penetrating matrigel also decreased from 442.6 ± 57.1 vs 376.4 ± 55.2 and 588.2 ± 70.1 vs 513.6 ± 68.5 after transfection compared with untreated group（P0.05） . Conclusion Down-regulation the expression of HBX can suppress invasive growth of human HCC cell line HepG2.215 in vitro.