摘要
目的 分析DNA甲基转移酶 1(Dnmt1)基因的真核表达质粒对人结肠癌细胞错配修复基因甲基化及其转录水平和微卫星不稳定性 (MSI)的影响。方法 分别构建含有人的正义Dnmt1(HMT)和反义Dnmt1(THM)的真核表达质粒 ,将其转染入结肠癌SW1116细胞 ,以Western印迹实验分析各组细胞Dnmt1蛋白的表达情况。定量PCR检测hMLH1、hMSH2基因的表达。甲基化特异性PCR分析hMLH1、hMSH2启动子区甲基化情况 ,银染法研究其对微卫星不稳定性的影响。结果 转染正义Dnmt1质粒的细胞hMLH1、hMSH2的启动子甲基化水平升高 ,基因mRNA表达降低。转染反义Dnmt1质粒可使细胞中hMSH2启动子呈非甲基化状态 ,基因表达增强。未发现转染正义和反义Dnmt1基因的SW1116细胞存在MSI的变化。结论 重组Dnmt1表达质粒可通过调控人结肠癌细胞中错配修复基因的甲基化影响基因的表达。
ObjectiveTo analyze the effect of eukaryotic plasmids containing sense or antisense DNA methyltransferase (Dnmt1) genes on the methylation status and transcription level of DNA mismatch repair (MMR) genes and microsatellite instability (MSI) in human colon cancer cell line.Methods Human colorectal cells of the SW1116 line were cultured. Recombinant plasmids containing sense Dnmt1 (HMT) or antisense Dnmt1 (THM) gene,pCMV-HMT and pCMV-THM,were constructed. Then pCMV-HMT,pCMV-THM,and pcMV blank plasmid were transfected into SW1116 cells respectively by using lipofectAMINE.The expression of Dnmt1 protein was examined by Western blotting.The transcription levels of hMLH1 and hMSH2 genes were detected by using real-time (RT-PCR). The status of methylation in promoters of hMLH1 and hMSH2 genes were examined with methylation specific PCR (MSP). The MSI of DNA in SW1116 cells was evaluated by silver-stained polyacrylamide gel electrophoresis. ResultsBoth the expressions of the hMLH1 and hMSH2 gene mRNAs were remarkably decreased in the SW1116-HMT cells in comparison with those in the untransfected cells. The expression of hMSH2 gene mRNA in the SW1116-THM cells was remarkably increased in comparison with that in the untransfected cells. No significant difference in the expressions of the hMLH1 and hMSH2 gene mRNAs was found between the SW1116 cells transfected with blank pCMV and the untransfected SW1116 cells. MSP showed that the methylation level in the regions of hMLH1 and hMSH2 promoters was remarkably increased in the SW1116 cells transfected with sense Dnmtl plasmid. However,in the SW1116 cells the hMSH2 promoter region was changed from partilly-methylated into de-methylated,and the hMLH1 promoter region remained non-methylated. MST test showed that extra bands indicating MSI were seen only in the D2S123 groups. ConclusionDnmt1 regulates the expression and methylation status of MMR genes and affects MSI in human colon cancer cell line SW1116.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2004年第12期1014-1017,共4页
National Medical Journal of China
基金
国家自然科学基金资助项目 (3 0 170 413 )
上海市教委"曙光计划"基金资助项目
