The Effect And Mechanism Of Epigenetic Regulation In Trichloroethylene-induced Hepatic Cytotoxicity

Trichlorethylene(TCE)is a widely used organic solvent in industrial production,occupational exposure and environmental exposure to TCE have become a seriouse health issue of general concern.Liver is one of the major organs in which TCE was metabolized and induced toxicity.It is of great practical significance to further explore the molecular mechanism of TCE-induced hepatotoxicity.Our previous studies have verified that SET was as a key mediator of TCE-induced hepatic cytotoxicity.Two microRNAs(miR-21a,miR-199b-5p)were also predicted regulating SET by directly binding to its 3'-UTR through a screening of various databases and quantitative PCR.Additionally,TCE could induce DNA damage and promote p53 expression in L-02 cells.Moreover,SET also suppresses the expression of p53 and aggravates the TCE-induced DNA damage.Based on these observations,we intend to explore the mechanism of SET-related upstream and downstream epigenetic regulation and its mechanism in TCE-induced hepatotoxicity.The research includes two parts as follow:Part 1:The interaction between miRNAs and SET,and their functionalities inTCE-induced hepatocyte apoptosis.Methods:Dual-luciferase reporter assay was used to verify the interaction between miRNAs(miR-21a,miR-199b-5p)and SET-3 'UTR.The expression of SET was evaluated by Western-blot analysis.Cell apoptosis was analyzed by flow cytometry.Results:The dual luciferase reporter assay and western blot analysis suggested that miR-199b-5p could suppress SET through directly binding to its 3'-UTR.Apoptotic assay indicated that after knockdown of miR-199b-5p in L-02 cells,the apoptotic rate was significantly decreased under the treatment of TCE.However,after overexpression of miR-199b-5p,the apoptotic remained unchangedrate under the treatment of TCE.Conclusion:miR-199b-5p can suppress SET by directly binding to SET-3 'UTR which further partially attenuated TCE-induced hepatocyte apoptosisPart 2:SET-mediated alteration of p53 through regulation of th H3K79me2 in TCE-induced DNA damageMethods:L-02 cells and SET-knockdown L-02 cells were treated with TCE for 24h.The H3K79me2 within the promoter region of p53 was measured by Chromatin immunoprecipitation-quantitative PCR(CHIP-qPCR).SiRNAs were synthesized to inhibit the H3K79 specific methyltransferase DotlL.The expression of p53 and H3K79me2 in L-02 cells transfected with DotlL-siRNA were analyzed by western blot.The DNA damage was evaluated by Single cell gel electrophoresis(SCGE).Results:The results indicated that SET suppressed H3K79me2 within the promoter region of p53 under the treatment of TCE.Further inhibition of H3K79 specific methyltransferase DOTlL verified that SET-mediated decreasing H3K79 di-methylation induced down-regulation of p53 and aggregated DNA damage.Conclusion:SET mediated decreased level of H3K79me2 within p53 promoter region and further down-regulated p53which aggravated the DNA damage induced by TCE.