Objective: To study the regulation of suppressor of cytokine signaling 3 (SOCS3) expression bythe brother of the regulator of the imprinted site (BORIS) in hepatocellular carcinoma cell.
Methods: The expression of mRNA in HCC cell lines was detected by real-time quantitative PCR (qRT-PCR). The expression of SOCS3 protein in knockdown and overexpression of HCC cell lines was tested by Western blot. The gene promoter methylation statusin the knockdown and overexpression of hepatocarcinoma cell lines was detected by using methylation specific PCR (MSP-PCR) method.The potential binding site of promoter region was found by UCSC database analysis.The enrichment of in promoter region in endogenous high expression of HCC cells was evaluated by using chromatin immunoprecipitation (ChIP)-qPCR (ChIP-qPCR).The promoter region histone methylation status in the knockdown and overexpression of HCC was detected by ChIP-qPCR.
Results: The expression of mRNA in hepatocellular carcinoma cells was higher and SOCS3 protein expression was down-regulated or up-regulated in the knockdown or overexpression of mRNA hepatocarcinoma cells,so has a positive regulatory effect on SOCS3 protein expression in hepatocarcinoma cells. MSP-PCR experiments showed that the promoter in SMMC-7721 and HepG2 cells was unmethylated and knockdown of did not change the methylation status; the promoter region of Huh7 cells was methylated; after overexpression of ,the promoter region was changed to an unmethylated state; the promoter was unmethylated in HCCLM3,overexpression of did not alter the methylation status. The ChIP-qPCR assay demonstrated that specifically binds to the promoter region in HCC cells with high expression of Histone methylation assay indicated that knockdown of reduced enrichment in the promoter region, with decreasing H3K4 me2 and increasing H3K27 me3 in the region of histone,whereas the overexpress in HCC cells showed the opposite situation.
Conclusion: BORIS plays a role of epigenetic regulationon gene promoter methylation and histone methylation,modulating the expression of SOCS3,and then involved in the development of hepatocellular carcinoma.
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