Modulation Effect Of SET On Histone Modification In Hepatocarcinoma Model Induced By Trichloroethylene

Trichloroethylene(TCE)is a chlorine-containing organic solvent occupational toxicant.TCE’s environment pollution problems caused by improper industrial production for many years have drawn increasing attention.EPA and IARC have identified TCE as a class I human carcinogen and the liver is the main important impaired organ for TCE metabolism,but the exact mechanism through which it causes hepatic impairment remains insufficient.Our preliminary research have found that SET(Patient SE translocation,SET)is a key protein for TCE-induced hepatocellular toxicity,which could cause down-regulation of histone H3K79me3 in the promoter region of p53 and promote the modification of H2AK9ac by inhibiting the expression of HDAC1 in hepatocyte exposure to TCE acutely.It has been indicated that abnormal histone modification may play a role in cancer.Up to now,there is no report regarding the association between histone post-translational modification and regulation of related gene expression in hepatic carcinoma models induced by chronic TCE exposure.This study intends to explore the expression of histone-related modifications in hepatic carcinoma induced by chronic TCE exposure in both cell and animal models,providing scientific clues and theoretical basis for revealing the mechanism underlying chronic hepatotoxicity in TCE.This study is mainly divided into the following three parts:1.Establishment and identification of B6C3F1 mice model of hepatic carcinoma induced by chronic trichloroethylene exposureMethods:B6C3F1 mice were divided into 3 groups(40 mice for each)and treated with different doses of TCE(500,1000 and 2000 mg/kg)by oral gavage for 5 times per week,56 weeks in total.Negative control(NC)were treated with corn oil(vector of TCE)and positive control(PC)were set at the same time.After the treatment,mouse liver specimens were isolated,identified by pathological analysis and used for calculating viscera coefficients and biochemical index.Results:The overall survival rate was 90.4%,and saw no significance difference between groups(P>0.05).Compared with vector control group,hepatic coefficients of high dose TCE group significantly increased(P<0.05).In the biochemical indicators,ALT and AST increased in a dose-effect and the lactate dehydrogenase(LDH)level increased in each dose TCE treatment group(P<0.05).Massive tumors were observed in liver tissues of treatment group,which were pathologically identified as massive hepatocellular carcinoma.The cancer rates in different groups were elevated in a dose-dependent manner:0(NC),5.26%(500 mg/kg),11.43%(1000 mg/kg),32.43%(2000 mg/kg)and 62.50%(PC)(P<0.001).Conclusion:We have successfully established a B6C3F1 mouse model of hepatic carcinoma induced by chronic TCE exposure.2.Establishment and preliminary identification of malignant transformation model induced by TCE long-term exposureMethods:Normal hepatocytes(L-02)and SET-deficient hepatocytes(SET-siRNA)were treated with 8 mmol/L(1/2 IC50)of TCE.TCE treatment was performed twice a week,and a soft agar colony formation assay was performed after 26 weeks of continuous exposureResults:Compared with the control group,L-02 cells and SET-siRNA cell of the exposure group were in a state of borders blur,in overlapping disorganized growth,and their colony formation rate both increased(P<0.01).Conclusion:L-02 cells and SET-siRNA cells exhibited a tendency of malignant transformation after 26 weeks of TCE exposure3.Histone modification and related protein expression changes in TCE-induced hepatic carcinoma modelMethods:Proteins were extracted from in vitro and in vivo models of hepatic carcinoma induced by TCE,and the expression levels of H3K79 methylation and acetylation,HDAC1,H2AK9 acetylation-related proteins were detected by Western blot.Results:In animal hepatic carcinoma model,the levels of SET and p53 increased in the middle and high dose groups(P<0.05),the level of H3K79me3 decreased,the level of HDAC1 decreased and the level of H2AK9ac increased(P<0.05)as compared to control group.In the cell model,compared with the control group,the level of H3K79me3 increased,the level of HDAC1 decreased and the level of H2AK9ac increased in L-02 cells,while the level of H3K79me3 decreased,the level of HDAC1 increased and the level of H2AK9ac decreased in SET-siRNA cells(P<0.05)after 26 weeks exposure of TCE;compared with the L-02 cells,the level of H3K79me3 increased in SET-siRNA cells of the exposure group(P<0.01).Conclusion:In the hepatic carcinoma induced by TCE,SET mediates a decrease in H3K79me3,in addition,SET may be promote H2AK9ac modification level by inhibiting HDAC1 expression.