人DNA MTase反义基因转染诱导E-钙黏附蛋白高表达

Demethylation in the promoter area by the antisense of human DNA MTase gene

目的 观察人DNAMTase反义基因转染后肝癌细胞系SMMC 772 1E 钙黏附蛋白表达的变化。方法 用基因重组技术构建包含人DNAMTase正、反义基因片段的真核表达载体 ;采用脂质体法将其转染入肝癌细胞系SMMC 772 1;采用RT PCR法观察DNAMTase基因mRNA、E 钙黏附蛋白基因mRNA表达变化 ;以甲基化特异的PCR法检测E 钙黏附蛋白基因启动子区域甲基化状态的改变 ;以免疫组化和流式细胞法检测E 钙黏附蛋白表达变化。结果 正、反义真核表达载体构建成功并转染入SMMC 772 1细胞 ,其中DNAMTase基因mRNA表达减低 ,E 钙黏附蛋白基因mRNA表达上调 ,E 钙黏附蛋白基因启动子区域呈现去甲基化状态 ,E 钙黏附蛋白表达上调。结论 抑制DNAMTase基因的表达可使肝癌SMMC 772 1细胞中E 钙黏附蛋白基因启动子区域去甲基化和表达增加 ;DNAMTase通过诱导启动子区域高甲基化 ,可使E

Objective To investigate the change in the expression of E Cadherin of human hepatocarcinoma cell line SMMC 7721 after transfection by antisense human DNA MTase gene. Methods DNA MTase gene eukaryotic expression vectors, including sense and antisense fragments, were constructed with recombinant technology and transfected into the hepatocarcinoma cell line SMMC 7721 with liposome DOTAP. The expression of DNA MTase gene mRNA and E Cadherin gene mRNA was examined with RT PCR and the expression of E Cadherin with immunohistochemical method and flow cytometry. The status of methylation in E Cadherin gene promoter area was examined with methylation specific PCR(MSP). Results The sense and antisense eukaryotic expression vectors were successfully constructed and then the constructed recombinant plasmids were successfully transfected into SMMC 7721 cell with liposome DOTAP. The expression of endogenous DNA MTase mRNA was obviously decreased with E Cadherin gene mRNA and its activity increased in the SMMC 7721 cell, which was tranfected with antisense DNA MTase gene fragment. Moreover, demethylation in the promoter area of E Cadherin gene was observed with MSP. Conclusion Demethylation in the promoter area and increasing mRNA level of E Cadherin gene can be induced by expression inhibition of DNA MTase gene of SMMC 7721 cell line.