Epigenetic ReguLation Of SET In TCE-induced Hepatic Cytotoxicity

Objective:Trichloroethylene(TCE) was widely used as an organic solvent in many industries because of the low cost. However, TCE has turned into a serious environmental issue by becoming one of the main ground water contaminations. TCE cou Ld be absorbed into human body by respiratory system and skin resorption. Liver is one of the major organs in which TCE was metabo Lized and attacked even resu Lted in hepatic failure or death for some severe cases. However, mechanisms of TCE-induced hepatic cytotoxicity still remains unclear. Studies showed that SET(protein phosphatase 2A inhibitor 2) was over expressed in Liver cancer cells and breast cancer cells. SET is an oncoprotein which involved in many key biological processes, such as cell apoptosis, transcription and chromosome replication etc. In our previous studies, we found out that SET mediated the TCE-induced hepatic cytotoxicity in L-02 cells, and analyzed the global methylation of whole genome in L-02 cells after exposure to TCE. In this study, we focused on studying the alterations of methylation on SET promoter and the mi RNAs which targeted to SET in TCE-induced hepatic cytotoxicity. Differential methylation on SET promoter in TCE-induced hepatic cytotoxicity. Methods: L-02 cells were exposed to TCE at different doses(0 mmol/L, 1 mmol/L, 2 mmol/L, 4 mmol/L, 8 mmol/L) for 24 h. Then the genomic DNA was extracted,and treated with Epi Tect® Bisulfite kit, the designed primers were used for DNA amplification, molecular cloning and sequencing. ResuLts and concLusions: Overall methylation of SET promoter was decreasing with the increasing concentration of TCE exposure in L-02 Liver cells. 73 differentially methylated sites were found with statistical significance(p<0.05) and 9 of which were known as transcription factor binding sites. Alterations of methylation in these transcription factor binding sites may induce the abnormal expression of SET.Those results explained and supported the up-regulation of SET protein in TCE treated L-02 liver ce Lls in an epigenetic view. Alterations of mi RNAs targeted to SET in TCE-induced hepatic cytotoxicity Methods: We predicted mi RNAs potentially targeted to SET by mi RNA-target prediction algorithms.RT-PCR was used to detect the expressions of the mi RNAs. Based on the results, only decreased mi RNAs were chosen for further study. The mi RNA over expressed L-02 cells were then constructed and verified in the study. ResuLts and concLusions: 9 mi RNAs were predicted targeting to SET by using mi RNA-target prediction algorithms, mi R-199 b, mi R-21 and mi R-23 a decreased were chosen to be over expressed respectively in L-02 cells. mi R-199 b, mi R-21 and mi R-23 a were verified respectively with 1300,3, 5 fo Lds up-regulation compared with contro L L-02 cells.L-02 cells with over expressed mi R-199 b, mi R-21 and mi R-23 a were constructed successfully respectively.