RNA干扰Dnmt1和Dnmt3b对小鼠胚胎成纤维细胞凋亡和基因组DNA甲基化水平的影响  

The Impact of Knockdown of Dnmt1 and Dnmt3b on the Apoptosis of Mouse Embryonic Fibroblast Cells and Global DNA Methylation Level

作　　者：吴梦华[1] 王巨龙[1] 张宇[1] 易建明[1] 于孟飞[2] 

机构地区：[1]华中农业大学动科动医学院,中国湖北武汉430070 [2]中南民族大学生命科学学院,中国湖北武汉430074

出　　处：《生命科学研究》2016年第1期1-7,共7页Life Science Research

基　　金：中央高校基本科研业务费专项资金资助(2013PY130);动物胚胎工程及分子育种湖北省重点实验室开放课题资助项目(2012ZD117)

摘　　要：DNA甲基转移酶1(DNMT1)负责DNA甲基化维持,DNA甲基转移酶3b(DNMT3b)主要负责DNA从头甲基化,在体细胞中同时干扰Dnmt1、Dnmt3b表达会给细胞带来何种影响还未见报道。实验以小鼠胚胎成纤维细胞为实验对象,用RNA干扰方法对Dnmt1和Dnmt3b进行敲低,研究分别干扰和同时干扰这两个基因对小鼠胚胎成纤维细胞凋亡、基因组甲基化水平的影响。研究发现,si RNA转染后24 h,Dnmt3b干扰组和Dnmt1+Dnmt3b同时干扰组细胞的增殖细胞核抗原(PCNA)mRNA水平显著降低(P<0.05);转染后48h,Dnmt3b单独干扰组、Dnmt1+Dnmt3b同时干扰组中细胞凋亡显著增加(P<0.05);Dnmt3b单独干扰组细胞基因组甲基化水平下降32%(P<0.01),Dnmt1+Dnmt3b同时干扰组细胞的基因组甲基化水平下降约44%(P<0.01)。结果表明,DNMT3b在小鼠胚胎成纤维细胞正常增殖、基因组甲基化水平的维持上具有重要作用。DNA Methyltransferase 1 （DNMT1） functions as a maintenance enzyme to ensure the methylation patterns to faithfully copy to progeny cells during DNA replication, while DNA methyhransferase 3b （DN- MT3b） mainly regulates the initial methylation for developing genomes. The effects of knockdown of Dnmtl and/or Dnmt3b on the changes of cellular apoptosis and global DNA methylation in mouse embryonic fibroblast cells （MEFs） were investigated using siRNA-mediated RNAi technology. The results demonstrated that the mRNA levels of proliferating cell nucleus antigen （PCNA） decreased significantly in both Dnmt3b singleknockdown cells and Dnmtl＋Dnmt3b doublenockdown cells （P〈0.05）. Moreover, at 48 h post-transfection, the rates of cells undergoing apoptosis in these two groups increased significantly（P〈0.05）. The global methylation level of Dnmt3b single-knockdown cells dropped 32% （P〈0.01）, whereas it decreased about 44% in the Dnmtl＋Dnmt3b double-knockdown group （P〈0.01）. These data suggested that DNMT3b played an im portant role in the maintenance of cellular proliferation and the global methylation states in MEFs.

关 键 词：DNA甲基转移酶1 DNA甲基转移酶3B DNA甲基化 RNA干扰 小鼠胚胎成纤维细胞 

分 类 号：Q255[生物学—细胞生物学]