The Function Of SET In Toxicity Of The Liver Cells Induced By Trichloroethylene And Its Epidemiologic Significance

Objectives:To identify the role of SET protein in the toxicity of liver cell’s induced by trichloroethylene (TCE), and provid necessary information for the possibility of SET protein as the biology marker of TCE occupational poisoning and revealing the mechanism of cytotoxic effect indued by TCE.Methods:This study is based on the former research of differentially expressed proteins induced by TCE, aimed at the SET protein, which is repoted as protein phosphatase 2A (PP2A) inhibitory with important biological functions. And lentivirus-mediated RNAi technology were used to establish the SET-silent stable L-02 liver cell line, Western Blot and fluorescence quantitative PCR were used to detect the expression level of SET in L-02 liver cells treated with different concertrations of TCE(0.25,0.5,1.0,2.0,4.0,8.0 mmol/L TCE) after different time durations (for 6 h,12 h and 24 h), and then cell proliferation, cell apoptosis, SET protein’s intracellular localization in both normal L-02 liver cells and SET-silent stable L-02 liver cell line treated with TCE were detected.Results:①Sequencing results showed that the SET-targeting shRNA lentiviral expression vectors were constructed successfully. Western Blot and fluorescence quantitative PCR were used to detect the effect of RNA interference against SET, and the results showed the psiRNA4 liver cell group was the best and stable, whose interference effect rate was over 90%.②CCK-8 assay results of L-02 liver cells treated with different concentrations (5,10,15,20,25,30,35,40 mmol/L)of TCE for 24 h showed that TCE significantly inhibited the L-02 liver cell proliferation in a concertration-effect relationship manner, and the half inhibitory concentration (IC50) was 15.95mmol/L.③Compared to the control group, both the mRNA and protein expression levels of SET in 6 h,12 h and 24 h treatment groups were upregulated, and the expression of SET in the 12 h treatment group was the highest in all groups;And PP2A activity test in 6 h,12 h and 24 h treatment groups were decreased, it confirmed indirectly the expression changes of SET through Western Blot and Fluorescence quantitative PCR in liver cells treated with TCE.④Compared with normal liver cells, the early apoptosis rate of the stable SET-silent liver cells was much higher, indicating that the inhibition of SET protein expression can promote liver cells apoptosis.The cell apoptosis rate increased with the TCE concertration’increasing.⑤Compared to normal liver cells, the amplification rate was slower in the stable SET-silent liver cells, and it is smaller, edge blurred.⑥SET protein was expressed in the nuclei of the liver cells, and the subcellular localization of SET in the liver cells was not changed after treatment with TCE.Conclusions:①SET-targeting shRNA lentiviral expression vectors and the stable SET-silent liver cell line were constructed successfully.②TCE is cytotoxicity to liver cells, and the the half inhibitory concentration (IC50) of liver cells treated with TCE for 24 h was 15.95 mmol/L.③CE can induce SET’s expression upregulation in the liver cells.④The amplification activity of the liver cells decresed after SET was slient.⑤SET protein could inhibit liver cells’apoptosis,and TCE could promote liver cells’apoptosis.⑥SET protein was expressed in the nuclei of the liver cells, and the treatment with TCE would not change the subcellular localization of SET in the liver cells.