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何靖炀, 刘秋英, 魏玲等. BORIS通过表观修饰对人肝癌细胞SOCS3表达的调控[J]. 四川大学学报(医学版), 2018, 49(1): 1-7.
引用本文: 何靖炀, 刘秋英, 魏玲等. BORIS通过表观修饰对人肝癌细胞SOCS3表达的调控[J]. 四川大学学报(医学版), 2018, 49(1): 1-7.
HE Jing-yang, LIU Qiu-ying, WEI Ling. et al. BORIS Regulates SOCS3 Expression Through Epigenetic Mechanisms in Human Hepatocellular Carcinoma Cells[J]. Journal of Sichuan University (Medical Sciences), 2018, 49(1): 1-7.
Citation: HE Jing-yang, LIU Qiu-ying, WEI Ling. et al. BORIS Regulates SOCS3 Expression Through Epigenetic Mechanisms in Human Hepatocellular Carcinoma Cells[J]. Journal of Sichuan University (Medical Sciences), 2018, 49(1): 1-7.

BORIS通过表观修饰对人肝癌细胞SOCS3表达的调控

BORIS Regulates SOCS3 Expression Through Epigenetic Mechanisms in Human Hepatocellular Carcinoma Cells

  • 摘要: 目的 探讨人肝癌细胞系中印记位点结合因子(BORIS)对细胞因子信号传导抑制因子-3(SOCS3)表达的调控方式。方法 通过实时荧光定量PCR(qRT-PCR)检测SOCS3 mRNA在肝癌细胞中的表达;Western blot检测敲低和过表达BORIS的肝癌细胞中SOCS3蛋白的表达;在敲低和过表达BORIS的肝癌细胞系中使用甲基化特异性PCR(MSP-PCR),检测SOCS3基因启动子区域甲基化状态;通过UCSC数据库分析,找到SOCS3基因启动子区潜在的BORIS结合位点,使用染色质免疫共沉淀(ChIP)-实时定量PCR(ChIP-qPCR)探索内源性高表达BORIS细胞中BORISSOCS3启动子区的富集状况;通过ChIP-qPCR检测敲低和过表达BORISSOCS3启动子区组蛋白甲基化状态。结果 在肝癌细胞中SOCS3 mRNA表达较高,在敲低或过表达BORIS mRNA肝癌细胞中,SOCS3蛋白表达相应下调或上调,即在肝癌细胞中BORIS以正向调控的方式调控SOCS3蛋白质的表达;MSP-PCR实验显示在SMMC-7721和HepG2细胞SOCS3启动子为非甲基化,敲低BORIS不改变甲基化状态,Huh7细胞的SOCS3启动子区为甲基化状态,过表达BORIS后,SOCS3启动子区转变为非甲基化状态,但在HCCLM3中SOCS3启动子为非甲基化,过表达BORIS不影响甲基化状态;ChIP-qPCR表明在内源性高表达BORIS的细胞中,BORIS特异性结合在SOCS3启动子区;组蛋白甲基化检测结果表明,敲低BORIS减少了BORIS在SOCS3启动子区富集,同时减少该区域H3K4 me2,增加H3K27 me3,而在过表达BORIS的细胞中呈现相反的结果。 结论 BORIS作为一个表观遗传调控因子调控SOCS3基因启动子区的甲基化状态和组蛋白甲基化修饰,影响SOCS3的表达,进而参与肝癌发生发展的过程。

     

    Abstract: 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 SOCS3 mRNA in HCC cell lines was detected by real-time quantitative PCR (qRT-PCR). The expression of SOCS3 protein in knockdown and overexpression BORIS of HCC cell lines was tested by Western blot. The SOCS3 gene promoter methylation statusin the knockdown and overexpression BORIS of hepatocarcinoma cell lines was detected by using methylation specific PCR (MSP-PCR) method.The potential BORIS binding site of SOCS3promoter region was found by UCSC database analysis.The enrichment of BORIS in SOCS3 promoter region in endogenous high expression BORIS of HCC cells was evaluated by using chromatin immunoprecipitation (ChIP)-qPCR (ChIP-qPCR).The SOCS3 promoter region histone methylation status in the knockdown and overexpression BORIS of HCC was detected by ChIP-qPCR. Results The expression of SOCS3 mRNA in hepatocellular carcinoma cells was higher and SOCS3 protein expression was down-regulated or up-regulated in the knockdown or overexpression of BORIS mRNA hepatocarcinoma cells, so BORIS has a positive regulatory effect on CM(155.3mmSOCS3 protein expression in hepatocarcinoma cells. MSP-PCR experiments showed that the SOCS3 promoter in SMMC-7721 and HepG2 cells was unmethylated and knockdown of BORIS did not change the methylation status; the SOCS3 promoter region of Huh7 cells was methylated; after overexpression of BORIS, the SOCS3 promoter region was changed to an unmethylated state; the SOCS3 promoter was unmethylated in HCCLM3, overexpression of BORIS did not alter the methylation status. The ChIP-qPCR assay demonstrated that BORIS specifically binds to the SOCS3 promoter region in HCC cells with high expression of BORIS. Histone methylation assay indicated that knockdown of BORIS reduced BORIS enrichment in the SOCS3 promoter region,with decreasing H3K4 me2 and increasing H3K27 me3 in the region of histone, whereas the overexpress BORIS in HCC cells showed the opposite situation. Conclusion BORIS plays a role of epigenetic regulationon SOCS3 gene promoter methylation and histone methylation, modulating the expression of SOCS3, and then involved in the development of hepatocellular carcinoma.

     

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