microRNA-182-5p在三氯乙烯肝毒性中的作用机制
发布时间：2019-06-26 10:26
【摘要】：目的:以三氯乙烯染毒B6C3F1雄性小鼠,筛选肝脏内差异表达的microRNA,在小鼠永生化肝细胞系BNL CL.2、小鼠肝癌细胞系Hepa1-6和人肝癌细胞系HepG2中进一步分析所筛选的miR-182-5p的DNA甲基化调控机制及其在三氯乙烯引起的细胞增殖中的作用,目的是了解三氯乙烯肝毒性的毒作用机制,为评估三氯乙烯对人肝脏的危害及其临床防治提供科学依据。方法:体内实验:将7周龄B6C3F1雄性小鼠随机分成4组,每组5只,以不同剂量三氯乙烯(0,100,500,1000 mg/kg b.w.)染毒5天,利用基因芯片筛选肝脏内差异表达的microRNA;以荧光定量PCR验证microRNA的表达,以荧光定量PCR和Western Blot检测预测靶基因mRNA和蛋白表达水平;以重亚硫酸盐测序方法检测microRNA启动子区DNA甲基化水平。细胞实验:用不同浓度三氯乙烯染毒处于对数生长期的BNL CL.2、Hepa1-6和HepG2细胞48小时,用MTT和平板克隆方法检测细胞增殖率和存活率;以流式细胞仪分析细胞周期及细胞凋亡;以碱性彗星实验检测对DNA损伤的影响;以miR-182-5p抑制物和类似物转染BNL CL.2和Hepa1-6细胞,用MTT和EdU标记实验检测对三氯乙烯染毒细胞的增殖影响;以甲基化酶抑制剂5-aza-2’-dC和组蛋白乙酰化酶抑制剂TSA处理细胞,检测对miR-182-5p表达的影响;用荧光定量PCR和Western Blot检测预测靶基因的mRNA和蛋白表达。结果:(1)1000mg/kg b.w.三氯乙烯染毒5天的小鼠肝脏中有8个miRNA表达上调,1个miRNA表达下降,其中miR-182-5p上调表达最为显著,并与三氯乙烯浓度有剂量关系;同时三氯乙烯可引起miR-182-5p基因启动子区低甲基化;三氯乙烯还导致mi R-182-5p预测靶基因Dnmt3a、Dnmt3b和Cited2等的mRNA表达水平下降,其中Cited2的mRNA和蛋白表达均降低。(2)与对照相比,非细胞毒性剂量(0.1和/或0.3mM)的三氯乙烯染毒48h后可引起BNL CL.2、Hepa1-6和HepG2细胞不同程度的增殖加快;平板克隆实验发现可使这三种细胞系的克隆形成率升高,存活率增加;流式细胞仪检测发现BNL CL.2和Hepa1-6细胞G1期阻滞减小;但对这三种细胞系不引起显著的细胞凋亡和DNA损伤。(3)在小鼠BNL CL.2、Hepa1-6和人HepG2细胞中,0.1和0.3mM三氯乙烯浓度下可引起miR-182-5p表达增加,并引起BNL CL.2细胞内预测靶基因Dnmt3a、Dnmt3b和Cited2等的mRNA表达水平下降,其中Cited2的蛋白表达水平也降低;miR-182-5p抑制物可逆转三氯乙烯引起的细胞增殖;5-aza-2’-dC处理可引起miR-182-5p表达增加。结论:三氯乙烯可能通过抑制肝脏中Dnmt3a和Dnmt3b等DNA甲基转移酶,降低miR-182-5p启动子区的DNA甲基化水平,促进miR-182-5p表达上升,并可能通过抑制其预测靶基因Cited2的表达促进细胞增殖,从而导致肝癌发生率升高。
[Abstract]:Objective: to screen the differentially expressed microRNA, in the liver of B6C3F1 male mice exposed to trichloroethylene and to further analyze the DNA methylation regulation mechanism of miR-182-5p and its role in the proliferation of cells induced by trichloroethylene in mouse immortalized BNL CL.2, mouse liver cancer cell line Hepa1-6 and human hepatocellular carcinoma cell line HepG2, in order to understand the toxic mechanism of trichloroethylene hepatotoxicity. It provides scientific basis for evaluating the harm of trichloroethylene to human liver and its clinical prevention and treatment. Methods: in vivo experiment: 7-week-old B6C3F1 male mice were randomly divided into 4 groups with 5 mice in each group with different doses of trichloroethylene (0, 100, 500, 1000 mg/kg b. W.) After 5 days of exposure, the differentially expressed microRNA; in the liver was screened by gene chip, the expression of microRNA was verified by fluorescence quantitative PCR, the expression level of target gene mRNA and protein was predicted by fluorescence quantitative PCR and Western Blot, and the DNA methylation level in microRNA promoter region was detected by bisulfite sequencing. Cell experiment: BNL CL.2,Hepa1-6 and HepG2 cells were exposed to different concentrations of trichloroethylene for 48 hours, the proliferation rate and survival rate were detected by MTT and plate cloning, the cell cycle and apoptosis were analyzed by flow cytometry, and the effects of alkaline comet assay on DNA damage were detected. BNL CL.2 and Hepa1-6 cells were transfected with miR-182-5p inhibitors and analogues, the proliferation of trichloroethylene cells was detected by MTT and EdU labeling assay, the effects of methylase inhibitor 5-aza-2'-dC and histone acetylase inhibitor TSA on the expression of miR-182-5p were detected, and the expression of mRNA and protein in target genes was predicted by fluorescence quantitative PCR and Western Blot. Results: (1) 1000mg/kg B. W. The expression of miRNA was up-regulated and the expression of miRNA was decreased in the liver of mice exposed to trichloroethylene for 5 days, and the up-regulated expression of miR-182-5p was the most significant, which was related to the concentration of trichloroethylene. At the same time, trichloroethylene could cause hypomethylation in the promoter region of miR-182-5p gene. Trichloroethylene also led to the decrease of mRNA expression of target genes Dnmt3a,Dnmt3b and Cited2 predicted by mi R 鈮，
本文编号：2506116
[Abstract]:Objective: to screen the differentially expressed microRNA, in the liver of B6C3F1 male mice exposed to trichloroethylene and to further analyze the DNA methylation regulation mechanism of miR-182-5p and its role in the proliferation of cells induced by trichloroethylene in mouse immortalized BNL CL.2, mouse liver cancer cell line Hepa1-6 and human hepatocellular carcinoma cell line HepG2, in order to understand the toxic mechanism of trichloroethylene hepatotoxicity. It provides scientific basis for evaluating the harm of trichloroethylene to human liver and its clinical prevention and treatment. Methods: in vivo experiment: 7-week-old B6C3F1 male mice were randomly divided into 4 groups with 5 mice in each group with different doses of trichloroethylene (0, 100, 500, 1000 mg/kg b. W.) After 5 days of exposure, the differentially expressed microRNA; in the liver was screened by gene chip, the expression of microRNA was verified by fluorescence quantitative PCR, the expression level of target gene mRNA and protein was predicted by fluorescence quantitative PCR and Western Blot, and the DNA methylation level in microRNA promoter region was detected by bisulfite sequencing. Cell experiment: BNL CL.2,Hepa1-6 and HepG2 cells were exposed to different concentrations of trichloroethylene for 48 hours, the proliferation rate and survival rate were detected by MTT and plate cloning, the cell cycle and apoptosis were analyzed by flow cytometry, and the effects of alkaline comet assay on DNA damage were detected. BNL CL.2 and Hepa1-6 cells were transfected with miR-182-5p inhibitors and analogues, the proliferation of trichloroethylene cells was detected by MTT and EdU labeling assay, the effects of methylase inhibitor 5-aza-2'-dC and histone acetylase inhibitor TSA on the expression of miR-182-5p were detected, and the expression of mRNA and protein in target genes was predicted by fluorescence quantitative PCR and Western Blot. Results: (1) 1000mg/kg B. W. The expression of miRNA was up-regulated and the expression of miRNA was decreased in the liver of mice exposed to trichloroethylene for 5 days, and the up-regulated expression of miR-182-5p was the most significant, which was related to the concentration of trichloroethylene. At the same time, trichloroethylene could cause hypomethylation in the promoter region of miR-182-5p gene. Trichloroethylene also led to the decrease of mRNA expression of target genes Dnmt3a,Dnmt3b and Cited2 predicted by mi R 鈮，
本文编号：2506116
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