组蛋白修饰在砷致DNA损伤修复中的调控机制研究

Arsenic is a confirmed human carcinogen. The arsenic pathogenic and carcinogenic mechanism is still unknown, which become the bottleneck problem for the complete control of endemic arsenism. As well as the major public health problem that is concerned by governments and scholars. Inhibition of DNA damage and repair has been recognized as the initial cause for arsenic carcinogenic function by many scholars, but the mechanism is not clear. Recent study showed that histone modifications play an important role in DNA damage repair process and in diseases development. In our study, based on the results of early stage research, the population exposed to endemic arsenic pollution from coal-burning in Guizhou province was selected as the research object, using real-time fluorescence quantitative polymerase chain reaction PCR(FQ-PCR), immunoprecipitation, western blotting, quantitative mass spectrometry to explore the characteristics of histone modifications in them, and to analyze the relationship between histone modifications and DNA damage and repair. Meanwhile, we build cell DNA double-strand breaks (DSB) model with gene recombination technology, and select the special histone modifications of arsenic with the cell model. And using the technology of point mutations in histone modifications or RNA interference knockout histone modification enzyme, to explore the histone modifications in the regulatory mechanism of DSB damage and repair caused by arsenic. The arsenic pathogenic and carcinogenic mechanism will be explored from new angle--histone modifications regulation in vivo and in vitro experiments system. And finding histone modifications biologic markers to provide a new ideas and new evidences for the prevention and therapy of arsenism.

砷是确认的人类致癌物，地方性砷中毒致病致癌机制至今不明是其未得以彻底控制的瓶颈问题，也是各国政府和学者高度关注的重大公共卫生问题。砷影响DNA损伤修复是砷致癌的始动因素已被众多学者认可，但机制尚不明确；新近研究推测，组蛋白修饰在DNA损伤修复过程及诱导疾病结局中起重要作用。本项目在课题组前期研究基础上，以贵州省燃煤污染型砷暴露人群为研究对象，采用荧光定量PCR、免疫沉淀、免疫印迹、定量质谱等方法探索砷暴露人群组蛋白修饰特征，分析其与DNA损伤修复的关系；采用基因重组方法构建细胞DNA双链断裂（DSB）模型，并应用该模型筛选砷特异性组蛋白修饰；采用组蛋白修饰特异位点突变技术或RNA干扰敲除组蛋白修饰酶技术，探索组蛋白修饰在砷致DSB损伤修复中的调控机制，从人群和体外实验两个体系、侧重组蛋白修饰调控新角度揭示砷的致病致癌机制，同时为寻找可能用于砷中毒防治的组蛋白修饰标志物性提供新思路和依据

地方性砷中毒致病机制不明，至今无特效治疗药物是限制其防治的瓶颈问题，也是各国政府和学者高度关注的重大公共卫生问题。研究表明，砷具有明确的遗传毒性，可通过抑制DNA的损伤修复，促进砷中毒的发生发展。新近研究显示，组蛋白修饰改变参与了DNA损伤修复的调控，但调控的机制尚不清楚。本研究侧重从人群和细胞两个体系，探讨组蛋白修饰在砷致DNA损伤中的调控作用，同时寻找可能用于砷中毒遗传损伤早期发现的组蛋白修饰标志，从表观遗传角度进一步揭示砷的分子毒作用机制，为地砷病的针对性预防和靶向治疗提供新思路和科学依据。. 研究发现：遗传损伤的难可逆性是砷中毒病情发生发展乃至癌变的重要原因之一；燃煤砷污染可导致砷中毒人群体内组蛋白H3K9me2、H3K36me3、H4K20me2、H3K18ac总体修饰水平异常改变；H3K9me2、H4K20me2、H3K18ac修饰水平与砷致DNA损伤呈负相关关系，H3K36me3修饰水平与砷致DNA损伤呈正相关关系；H3K9me2、H3K36me3、H3K18ac修饰水平的改变是砷中毒发生的早期事件，其改变仅与砷中毒的发生有关，与砷中毒严重程度无关，其有望作为组蛋白修饰标志物用于砷中毒的防治。染砷HaCaT细胞H3K9me2、H3K36me3、H4K20me1、H4K20me2总体修饰水平与DNA损伤水平均呈负相关关系；MPG、PARP1、XRCC1、DDB2、XPC、BRCA1, RAD51B，MGMT 是砷相关的DNA损伤修复靶基因，NaAsO2可能通过抑制其mRNA的转录表达，使细胞DNA损伤修复受阻；在砷的作用下，H3K9me2修饰在DNA损伤修复基因的启动子区能特异性结合增加，调控MPG、PARP1、XRCC1、DDB2、XPC、BRCA1, RAD51B，MGMT基因mRNA转录抑制，抑制砷导致的DNA损伤修复。人群和细胞研究均表明，砷可通过影响组蛋白甲基转移酶PR-Set7的表达调控H4K20me1修饰水平降低，进而抑制碱基切除修复基因PARP1、MPG、XRCC1 mRNA的转录表达，从而降低DNA损伤修复能力，导致DNA损伤加重，从而参与砷中毒皮肤病变的发生发展。根据本研究获得的特异性组蛋白修饰，筛选可逆转组蛋白修饰的药物，用于地砷病的防治中，对控制砷中毒危害具有重要的科学意义和应用价值.