三氯乙烯对B6C3F1雄性小鼠肝脏和肾脏中细胞增殖相关基因表达和DNA甲基化的影响

Cell proliferation-related mRNA expression and DNA methylation changes induced by trichloroethylene in the liver and kidney of B6C3F1 male mice

目的:三氯乙烯(TCE)是环境中广泛存在的工业污染物,可引起小鼠肝癌,但对肾癌发生未见显著影响。本研究通过检测TCE对小鼠肝脏和肾脏细胞增殖和DNA甲基化调控相关基因表达以及对DNA甲基化的影响,探讨TCE引起小鼠肝癌的分子机制。方法:将6周龄B6C3F1雄性小鼠随机分成3组,每组4只,分别以0、500和1 000 mg/kg剂量的TCE连续灌胃5 d。以荧光定量PCR方法检测TCE染毒小鼠肝脏、肾脏中与细胞增殖以及DNA甲基化调控相关基因的m RNA表达水平,以结合重亚硫酸盐的限制性内切酶方法检测Cdkn1a启动子区和重复序列的DNA甲基化水平。结果:与对照组相比,TCE可引起小鼠肝脏中细胞增殖相关基因Cdkn1a、Jun和Mki67的m RNA水平显著升高(P均<0.05),且呈剂量反应关系。同时1 000 mg/kg TCE染毒小鼠肝脏中主要DNA甲基化调控基因Dnmt3a、Dnmt3b和Tet2的m RNA水平降低(P均<0.05),Uhrf1 m RNA的表达升高(P均<0.05)。TCE染毒还导致肝脏内Cdkn1a启动子区的DNA甲基化水平降低,但对肾脏中相关基因及DNA甲基化水平无显著影响。结论:TCE引起的细胞增殖相关基因表达升高及DNA甲基化异常可能在其促进小鼠肝癌发生中起重要作用。

OBJECTIVE：Trichloroethylene（TCE）,a common industrial contaminant in the environment,could induce hepatocellular carcinoma but not kidney cancer in mice.This study aimed to understand the molecular mechanisms of TCE hepatocarcinogenicity by examining the effect of TCE on proliferation-related mRNA expression and DNA methylation in mouse liver and kidney.METHODS：B6C3F1 male mice aged six weeks were randomly divided into 3 groups,with 4 mice in each group.Mice were fed with TCE at 0,500 and 1 000 mg/kg for 5 days.Using qPCR,we examined the mRNA expression of genes involved in cell proliferation and in the regulation of DNA methylation in TCE-exposed mouse liver and kidney.The promoter methylation status of Cdknla and the DNA methylation level of repetitive sequences were detected by combined bisulfite restriction analysis.RESULTS：Compared with control,TCE increased the mRNA expression of Cdknla,Jun and Mki67 in a dose-dependent manner in mouse liver.Moreover,in mouse treated with TCE at 1000mg/kg,the mRNA expression of a number of key DNA methylation regulation genes including Dnmt3 a,Dnmt3b and Tet2 were down-regulated,while the mRNA expression level of Uhrf1 was up-regulated（all P〈0.05）.TCE also induced hypomethylation of the promoter region of Cdknla in mouse hepatocytes.However,TCE should no detectable effects on mRNA expression and DNA methylation in mouse kidney.CONCLUSION：Alterations in the mRNA expression of proliferationrelated genes and DNA methylation changes might be crucial to TCE-induced hepatocarcinogenesis in mice.