He Jing-Yang, Liu Qiu-Ying, Wei Ling, Liu Zhong-Jian, Huang Yuan, Yu Xiao-Qin, Li Bo, Qin Yang
Department of Biochemistry and Molecular Biology,West China School of Preclinical and Forensic Medicine,Sichuan University,Chengdu 610041,China.
Department of Liver and Liver Transplantation Center,West China Hospital,Sichuan University,Chengdu 610041,China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2018 Jan;49(1):1-7.
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.
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.
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.
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.
研究印记位点调控因子的兄弟（BORIS）对肝癌细胞中细胞因子信号转导抑制因子3（SOCS3）表达的调控作用。
采用实时定量聚合酶链反应（qRT-PCR）检测肝癌细胞系中mRNA的表达。通过蛋白质免疫印迹法检测肝癌细胞系中SOCS3蛋白在基因敲低和过表达后的表达情况。运用甲基化特异性聚合酶链反应（MSP-PCR）方法检测肝癌细胞系在基因敲低和过表达后基因启动子的甲基化状态。通过UCSC数据库分析寻找启动子区域的潜在结合位点。利用染色质免疫沉淀-qPCR（ChIP-qPCR）评估肝癌细胞内源性高表达时启动子区域的富集情况。通过ChIP-qPCR检测肝癌细胞在基因敲低和过表达后启动子区域组蛋白甲基化状态。
肝癌细胞中mRNA的表达较高，在mRNA敲低或过表达的肝癌细胞中，SOCS3蛋白表达下调或上调，因此对肝癌细胞中SOCS3蛋白表达具有正向调控作用。MSP-PCR实验表明，SMMC-7721和HepG2细胞中的启动子未甲基化，敲低后甲基化状态未改变；Huh7细胞的启动子区域甲基化；过表达后，启动子区域变为未甲基化状态；HCCLM3中的启动子未甲基化，过表达未改变甲基化状态。ChIP-qPCR分析表明，在高表达的肝癌细胞中特异性结合启动子区域。组蛋白甲基化分析表明，敲低可减少启动子区域的富集，组蛋白区域的H3K4 me2减少，H3K27 me3增加，而肝癌细胞中的过表达则呈现相反情况。
BORIS在基因启动子甲基化和组蛋白甲基化方面发挥表观遗传调控作用，调节SOCS3的表达，进而参与肝癌的发生发展。
