USP22/SIRT1/Bmi-1通过调节组蛋白泛素化影响砷诱导的肝细胞恶性转化过程

Arsenic has been classified as a definitive human carcinogen, but the mechanisms by which arsenic induces carcinogenesis are not fully elucidated. Recently, it is reported that USP22, SIRT1 and Bmi-1 regulate the gene expression via catalyzing histone ubiquitination.In our previous study, we found that the levels of USP22, SIRT1 and Bmi-1 protein increased, and the levels of ubH2A and ubH2B decreased in L-O2-As, a malignantly transformed variant of L-O2, that emerged from long-term exposure to arsenic. Thus, we proposed a hypothesis that USP22/SIRT1/Bmi-1 might be involved in arsenic induced carcinogenesis via regulating histone ubiquitination. So, in this proposal, we will first investigate the association between arsenic exposure and global histone ubiquitination; and then will use human liver L-O2 cells and L-O2-As as study models and analyze whether USP22/SIRT1/Bmi-1 are involved in the malignant transformation induced by arsenic using immunoprecipitation, western blot, chromatin immunoprecipitation and real time quantitative PCR. The study will provide theory basis and experiment data for elucidating the mechanisms of arsenic carcinogenesis and prevention and cure of arsenicalism.

确定致癌物砷的致癌作用机制尚未完全阐明。近来研究表明,USP22、SIRT1和Bmi-1可通过调节组蛋白泛素化水平而调控基因表达。本课题组发现，在具有恶性表型转化肝细胞株L-O2-As中，USP22、SIRT1和Bmi-1表达增加，ubH2A和ubH2B整体水平降低。因此，我们推测USP22、SIRT1和Bmi-1可能通过调节组蛋白泛素化水平而影响砷诱导的恶性转化过程。为此，我们首先通过分子流行病学调查证实ubH2A和ubH2B水平与砷暴露的关系；然后采用正常肝细胞株L-O2及本课题组建立的砷诱导恶性转化肝细胞株L-O2-As作为研究模型，采用免疫沉淀、western blot、CHIP及实时定量PCR 等方法探索USP22、SIRT1和Bmi-1是否通过调节组蛋白泛素化水平参与了砷诱导的肝细胞恶性转化过程，为深入理解砷致癌作用机制提供实验依据、为慢性砷中毒的防治提供理论基础。

确定致癌物砷的致癌作用机制尚未完全阐明。在本研究中，我们检测了几种人群外周血白细胞组蛋白修饰水平与砷暴露的关系；然后在细胞水平探讨了USP22、Sirt1和Bmi-1是否通过调节组蛋白泛素化水平参与了砷诱导的肝细胞恶性转化过程。本课题组建立的斑点杂交方法可应用于检测人群外周血白细胞中组蛋白修饰水平。饮水砷暴露可改变人群外周血白细胞中H2BK120Ac、H2BK120Ub及H3K4Me3、H3K79Me3和H3S10P水平，且这几种组蛋白修饰组合可更好地预测砷暴露的水平。砷可以通过诱导USP22表达来降低H2AK119Ub和H2BK120Ub在cyclin D2、hTERT、NAMPT和LDHA基因启动子区的富集，从而促进cyclin D2等4种c-myc靶基因的表达；砷也可以通过诱导Bmi-1表达来增加H2AK119Ub在抑癌基因p16、p21和PTEN启动子区的富集，从而降低p16等3种抑癌基因的表达；砷还可以通过诱导Sirt1表达来降低H3K9Ac和H3K27Ac在抑癌基因p16和p53启动子区的富集，从而抑制p16和p53的表达，进而促进肝细胞增殖或诱导肝细胞发生恶性转化。该研究为深入理解砷致癌作用机制以及慢性砷中毒的防治提供了理论和实验依据。