microRNA-182-5p在三氯乙烯肝毒性中的作用機(jī)制
發(fā)布時(shí)間：2019-06-26 10:26
【摘要】：目的:以三氯乙烯染毒B6C3F1雄性小鼠,篩選肝臟內(nèi)差異表達(dá)的microRNA,在小鼠永生化肝細(xì)胞系BNL CL.2、小鼠肝癌細(xì)胞系Hepa1-6和人肝癌細(xì)胞系HepG2中進(jìn)一步分析所篩選的miR-182-5p的DNA甲基化調(diào)控機(jī)制及其在三氯乙烯引起的細(xì)胞增殖中的作用,目的是了解三氯乙烯肝毒性的毒作用機(jī)制,為評(píng)估三氯乙烯對(duì)人肝臟的危害及其臨床防治提供科學(xué)依據(jù)。方法:體內(nèi)實(shí)驗(yàn):將7周齡B6C3F1雄性小鼠隨機(jī)分成4組,每組5只,以不同劑量三氯乙烯(0,100,500,1000 mg/kg b.w.)染毒5天,利用基因芯片篩選肝臟內(nèi)差異表達(dá)的microRNA;以熒光定量PCR驗(yàn)證microRNA的表達(dá),以熒光定量PCR和Western Blot檢測(cè)預(yù)測(cè)靶基因mRNA和蛋白表達(dá)水平;以重亞硫酸鹽測(cè)序方法檢測(cè)microRNA啟動(dòng)子區(qū)DNA甲基化水平。細(xì)胞實(shí)驗(yàn):用不同濃度三氯乙烯染毒處于對(duì)數(shù)生長(zhǎng)期的BNL CL.2、Hepa1-6和HepG2細(xì)胞48小時(shí),用MTT和平板克隆方法檢測(cè)細(xì)胞增殖率和存活率;以流式細(xì)胞儀分析細(xì)胞周期及細(xì)胞凋亡;以堿性彗星實(shí)驗(yàn)檢測(cè)對(duì)DNA損傷的影響;以miR-182-5p抑制物和類似物轉(zhuǎn)染BNL CL.2和Hepa1-6細(xì)胞,用MTT和EdU標(biāo)記實(shí)驗(yàn)檢測(cè)對(duì)三氯乙烯染毒細(xì)胞的增殖影響;以甲基化酶抑制劑5-aza-2’-dC和組蛋白乙�；敢种苿㏕SA處理細(xì)胞,檢測(cè)對(duì)miR-182-5p表達(dá)的影響;用熒光定量PCR和Western Blot檢測(cè)預(yù)測(cè)靶基因的mRNA和蛋白表達(dá)。結(jié)果:(1)1000mg/kg b.w.三氯乙烯染毒5天的小鼠肝臟中有8個(gè)miRNA表達(dá)上調(diào),1個(gè)miRNA表達(dá)下降,其中miR-182-5p上調(diào)表達(dá)最為顯著,并與三氯乙烯濃度有劑量關(guān)系;同時(shí)三氯乙烯可引起miR-182-5p基因啟動(dòng)子區(qū)低甲基化;三氯乙烯還導(dǎo)致mi R-182-5p預(yù)測(cè)靶基因Dnmt3a、Dnmt3b和Cited2等的mRNA表達(dá)水平下降,其中Cited2的mRNA和蛋白表達(dá)均降低。(2)與對(duì)照相比,非細(xì)胞毒性劑量(0.1和/或0.3mM)的三氯乙烯染毒48h后可引起B(yǎng)NL CL.2、Hepa1-6和HepG2細(xì)胞不同程度的增殖加快;平板克隆實(shí)驗(yàn)發(fā)現(xiàn)可使這三種細(xì)胞系的克隆形成率升高,存活率增加;流式細(xì)胞儀檢測(cè)發(fā)現(xiàn)BNL CL.2和Hepa1-6細(xì)胞G1期阻滯減小;但對(duì)這三種細(xì)胞系不引起顯著的細(xì)胞凋亡和DNA損傷。(3)在小鼠BNL CL.2、Hepa1-6和人HepG2細(xì)胞中,0.1和0.3mM三氯乙烯濃度下可引起miR-182-5p表達(dá)增加,并引起B(yǎng)NL CL.2細(xì)胞內(nèi)預(yù)測(cè)靶基因Dnmt3a、Dnmt3b和Cited2等的mRNA表達(dá)水平下降,其中Cited2的蛋白表達(dá)水平也降低;miR-182-5p抑制物可逆轉(zhuǎn)三氯乙烯引起的細(xì)胞增殖;5-aza-2’-dC處理可引起miR-182-5p表達(dá)增加。結(jié)論:三氯乙烯可能通過抑制肝臟中Dnmt3a和Dnmt3b等DNA甲基轉(zhuǎn)移酶,降低miR-182-5p啟動(dòng)子區(qū)的DNA甲基化水平,促進(jìn)miR-182-5p表達(dá)上升,并可能通過抑制其預(yù)測(cè)靶基因Cited2的表達(dá)促進(jìn)細(xì)胞增殖,從而導(dǎo)致肝癌發(fā)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，
本文編號(hào)：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，
本文編號(hào)：2506116
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