| Objective:We examined the mi RNA expression profiles in the liver of B6C3F1 male mice exposed to trichloroethylene(TCE). The differentially expressed miR-182-5p was further studied in mice immortalized liver cell line(BNL CL.2), mouse hepatoma cell lines(Hepa1-6) and human hepatoma cell line(HepG2), with emphaisises on its expression regulated by DNA methylation and on its role in TCE-induced cell proliferation. This study is useful for understanding the mechanisms of TCE hepatotoxicity, and can provide a scientific basis for prevention and treatment of hepatocellular carcinoma caused by trichloroethylene.Methods:In vivo experiments: Male B6C3F1 mice aged 7weeks were randomly divided into 4 groups, with 5 mice in each group. The mice were exposed to TCE at different dose levels(0,100,500 and 1000 mg/kg b.w.) for 5days. Using microRNA microarray, we examined differentially expressed microRNAs in the mice liver. microRNA expression was validated by RT-qPCR; mRNA and protein expression were examined by RT-qPCR and western blotting. Bisulfite sequencing was used to detect the DNA methylation status of microRNA promoter regions.In vitro experiments: BNL CL.2, Hepa1-6 and HepG2 cell lines in exponential growth phase were treated with TCE at different dose levels for 48 hours. Cell proliferation and survival rate were detected by MTT and colony formation assay. Using flow cytometry, we analyzed cell cycle and cell apoptosis. Alkaline comet assay was used to detect DNA damage. MTT and EdU labeled assay were used to detect cell proliferation in BNL CL.2 and Hepa1-6 cells exposed to TCE with presence or not of miR-182-5p inhibitor or mimics. In addition, we examined miR-182-5p expression in cells treated with DNA methyltransferase inhibitor 5-aza-2’-dC or histone deacetylase inhibitor TSA. For predicted target genes, RT-qPCR and western blotting were used to examine mRNA and protein expressionResults:(1)There were 8 microRNAs up-regulated and 1 microRNA down-regulated in the mice liver treated with TCE at a dose of 1000mg/kg b.w.. miR-182-5p was the most significantly up-regulated miRNA, and there is a dose-response relationship between miR-182-5p expression and increasing doses of TCE. Meanwhile, TCE could cause DNA hypomethylation in the promoter regions of miR-182-5p. The mRNA expression levels of predicted miR-182-5p target genes including Dnmt3a、Dnmt3b and Cited2 were downregulated. The protein expression level of Cited2 was also decreased.(2)Compared with controls, TCE treatment for 48 h at non-cytotoxic doses(0.1mM and/or 0.3m M) promoted cell proliferation in BNL CL.2, Hepa1-6 and HepG2 cells. TCE at non-cytotoxic dose levels also increased cell survival rate in the three cell lines. G1 phase block was diminished in BNL CL.2 and Hepa1-6 cells treated with TCE at 0.3 and 0.1mM respectively. No observable apoptosis and DNA damage was found in the three cell lines treated with TCE at non-cytotoxic dose levels.(3)In BNL CL.2, Hepa1-6 and Hep G2 cells, TCE at 0.1and /or 0.3 mM increased the miR-182-5p expression. The mRNA expression levels of predicted mi R-182-5p target genes including Dnmt3 a and Dnmt3 b were downregulated. Both mRNA and protein expression levels of Cited2 were downregulated. On the other hand, miR-182-5p inhibitor aborted TCE-induced cell proliferation. In addition, miR-182-5p was up-regulated in all the three cell lines treated with 5-aza-2’-dC.Conclusion:Our data indicates that TCE exposure down-regulates Dnmt3 a and Dnmt3 b, which could result in hypomethylation of the promoter region of miR-182-5p, leading to miR-182-5p overexpression. Up-regulation of miR-182-5p in turn inhibits Cited2, promotes cell proliferation, and contributes to TCE hepatocarcinogenesis. |
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