DNMT1 siRNA稳定表达载体的构建及其沉默效率的评价  

Construction of DNMT1 siRNA stable expressing vector and evaluation of its silenced efficiency in blocking gene expression

作　　者：樊红[1] 许军[1] 吴守伟[2] 赵主江[1] 张建琼[1] 谢维[1] 

机构地区：[1]东南大学遗传学研究中心,发育与疾病相关基因教育部重点实验室,江苏省基因诊断与治疗医学重点实验室,南京210009 [2]蚌埠医学院生物学教研室

出　　处：《中华医学遗传学杂志》2005年第2期142-145,共4页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金(30470950);教育部高校青年教师奖基金(2001/182)~~

摘　　要：目的　构建能在肝癌细胞系SMMC- 772 1中稳定表达的高效率DNMT1的RNA干扰载体。方法　合成特异性干扰DNMT1的小分子干扰RNA (small interfering RNA,si RNA )分子,并与p SUPER- GFP载体连接。脂质体转染SMMC- 772 1细胞,并以G4 18筛选稳定表达细胞系。应用逆转录-聚合酶链反应和Western印迹方法对细胞系中DNMT1的RNA表达水平和蛋白表达水平进行了分析。应用甲基化聚合酶链反应方法分析该细胞系中甲基化的基因E-钙粘着蛋白的甲基化状态。结果　转染DNMT1沉默载体的SMMC- 772 1细胞中DNMT1的m RNA表达量为对照载体p SU PER转染SMMC-772 1细胞的4 3% ,而前者中DNMT1蛋白表达水平小于后者的10 %。DNMT1的si RNA沉默效率大于90 % ,所构建的DNMT1的si RNA有较高的沉默效率。DNMT1的表达抑制使E-钙粘着蛋白基因启动子区出现去甲基化。结论　成功构建了能稳定表达的高效率DNMT1的RNA干扰载体,但目的基因沉默后其RNA表达水平与蛋白质表达水平表现不一致,评价si RNA干扰作用时应以目的基因相应蛋白质的表达水平为准。Objective To construct the specific stable expression and high efficiency small interfering RNA(siRNA) expression vector that can block DNMT1 gene function. Methods Using vector-based RNA interference technique, the authors constructed a vector to transcribe functional short interfering RNA (RNAi). After transfection by lipofectmine TM reagent, the treated cells were selected by G418. The expression levels of RNA and protein of DNMT1 were analyzed by reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting. The status of methylation of E-cadherin was analyzed by methylation-specific PCR(MSP). Results The expression level of endogenous DNMT1 mRNA in transfected SMMC-7721 cell lines with DNMT1 RNAi construct was 43% less than that in control cell 7721-pSU cell lines. The protein level in the former was about 10% less than that in the latter. The efficiency of the siRNA of DNMT1 was found to be higher than 90%. Demethylation of promoter of E-cadherin was obtained due to the inhibition of DNMT1. Conclusion DNMT1 siRNA stable expressing vector was obtained by gene-recombined technology. There was no complete sameness between the levels of protein and RNA in gene silenced cell lines. The efficiency of the siRNA should be confirmed by Western-blotting.

关 键 词：DNMT1 SIRNA 稳定表达载体 SMMC-7721细胞 Western印迹方法 逆转录-聚合酶链反应 E-钙粘着蛋白 蛋白表达水平 MRNA表达量 RNA干扰作用 基因启动子区 肝癌细胞系 脂质体转染 甲基化状态 RNA沉默 蛋白质表达 方法分析 去甲基化 

分 类 号：R73-3[医药卫生—肿瘤]