pSi2．1-G9a-siRNA诱导人胆管癌细胞RASSF1A基因脱甲基化

Demethylation of RASSF1A gene induced by pSi2. 1-G9a-siRNA plasmid in cholangiocarcinoma cells

目的分析组蛋白甲基转移酶G9a特异性siRNA真核表达质粒psi2．1．G9a．siRNA对人胆管癌细胞株QBC939中RASSF1A基因启动子甲基化及其表达的影响，探讨组蛋白甲基化和DNA甲基化的关系。方法构建pSi2，1．G9a—siRNA真核表达质粒并转染QBC939细胞，甲基化特异性PCR（MS-PCR）检测RASSFlA启动子甲基化状态的改变，逆转录-聚合酶链反应（RT．PCR）观察RASSFlAmRNA水平变化，Westernblot检测RASSFlA蛋白表达。结果转染pSi2．1-G9a-siR-NA后，QBC939细胞中RASSFlA启动子由甲基化状态转变为非甲基化状态；RT—PCR和Westernblot结果显示转染组细胞中分别出现RASSFlA基因的DNA条带（280bp）和蛋白质条带（40×10^3），而转染前没有相应条带出现。结论pSi2．1-G9a-siRNA质粒能够诱导QBC939中RASSFlA启动子去甲基化并恢复表达，提示DNA甲基化在一定程度上受组蛋白甲基化的调控。

Objective To explore the probable mechanism of DNA methylation modulation by histone methylation through studying the effect of pSi2. I-Gga-siRNA plasmid on the methylation status in the promoter region of RASSF1A gene and its expression in human cholangiocarcinoma cell,line QBC939. Methods pSi2.1-G9a-siRNA plasmid was eonstnleted and transfected into QBC939 cells. Methylation status in the promoter legion of RASSFI A gene was detected by methylation specific PCR （ MS-PCR）, and the expression of RASSF1A mRNA and protein was observed by RT-PCR and Western blot respectively. Results After transfected with psi2.1-G9a-siRNA plasmid,methylaiton status in the promoter region of RASSF1A gene was reversed from methylation to un-methylation. A 280 bp DNA band that presented RASSF1A expression at transcriptional level and a 40 kD protein band that presented RASSF1A expression at protein level were detected by RT-PCR and Western blot respectively in the experimental group,but no corresponding bands were found in the control group. Conclusion Demethylation in the promoter region and re-expression of RASSF1A induced by pSi2. 1-G9a-siRNA plasmid in QBC2939 suggest that DNA methylation may be modulated to some extent by histone methylation.