HCV非结构蛋白3反式激活基因6转染肝癌细胞的基因表达谱芯片分析

Screening of genes differentially expressed in HepG2 cells transfected with HCV NS3TP6 by microarray assay

目的:为了研究NS3TP6可能的分子生物学功能,我们应用基因芯片技术,检测丙型肝炎病毒(HCV)非结构蛋白3(NS3)反式激活基因NS3TP6的表达对肝母细胞瘤细胞HepG2基因表达谱的影响.方法:以分子生物学技术构建NS3TP6的真核表达载体pcDNA3.1(-)-NS3TP6,以表达质粒pcDNA3.1(-)-NS3TP6转染HepG2细胞,以空载体pcDNA3.1(-)为平行对照,制备转染后的细胞裂解液,提取mRNA.应用基因表达谱芯片技术对差异表达mRNA进行检测和分析.结果:HepG2细胞经转染NS3TP6后,有7条基因表达增强,38条基因表达降低.结论:应用基因表达谱芯片成功筛选了NS3TP6的反式调节基因,为进一步阐明NS3TP6的反式激活作用及免疫调节机制提供了新的依据.

AIM： To study the difference in gene expression in human hepatoblastoma cell line HepG2 cells transfected with HCV NS3TP6-expressing plasmid and to further elucidate its molecular biological mechanism. METHODS： Sequence specific primers were designed and synthesized and the NS3TP6-coding DNA fragment was amplified with polymerase chain reaction （ PCR ） technique using pBRTM3011 plasmid containing the full length of HCV-H cD- NA as the template. The expression vector of pcDNA3. 1 （ - ）-NS3TP6 was constructed by routine molecular biological methods, cDNA microarray technology was employed to detect the mRNA from the HepG2 cells transfected respectively with pcDNA3.1 （ - ）-NS3TP6 and pcDNA3.1 （ - ） using lipofectamine. RESULTS： The expression vector was constructed and confirmed by restriction enzyme digestion and DNA sequencing analysis. The expression of NS3TP6 protein was confirmed by Western blot hybridization with single chain variable region （scFv） antibody. High quality mRNA and cDNA were prepared and successful microarray screening was conducted. The scanning results indicated that among the 1152 genes which were obtained from gene expression profile analysis, 45 were different, of which 7 genes were up-regulated and 38 genes were down-regula- ted in NS3TP6-expressing HepG2 cells. CONCLUSION： cDNA microarray technology is successfully used to screen the genes differentially expressed in NS3TP6-expressing HepG2 cells, which brings some new clues for the study of the molecular mechanism involved in the HCV infection and hepatocellular carcinoma development induced by HCV NS3TP6 protein.