TET2介导STAT3低甲基化在苯造血毒性中的作用和机制

Prevention and treatment of hematopoietic toxicity focus on elucidating the mechanism and looking for early biomarkers. Our previous reports found that Signal Transducer and Activator of Transcription 3 (STAT3) hypomethylation participated in benzene hematopoietic toxicity, but the mechanism was still unclear. Ten-eleven translocation 2 (TET2), as hematopoiesis regulation proteins, can regulate DNA demethylation by hydroxylating 5-methylcytosine to 5-hydroxymethylcytosine. Therefore, we hypothesize that TET2 may be a key molecule of benzene hematopoietic toxicity, through mediating STAT3 methylation to regulate the renewal and differentiation of hematopoietic stem/progenitor cell, resulting in hematopoietic toxicity of benzene. Our group has been successfully established defined culture conditions for directing human induced pluripotent stem cells differentiation toward human hematopoietic stem/progenitor cells (hHSPCs). In order to reveal the effect and mechanism of STAT3 hypomethylation mediated by TET2 involved in benzene hematopoietic toxicity, the research was designed to explore the regularity of TET2 expression and STAT3 hypomethylation and to monitor the renewal and differentiation of hHSPCs disturbed by 1,4-BQ using high content screening (HCS), PCR-fluorescence probe, sequencing, methylation-specific PCR and RNAi. The correlations and dose-effect relationships of benzene exposure, the relative expression of TET2 and STAT3 and hemogram were analyzed based on a cohort of benzene exposure population. Taken together, this study is to clarify the effect and mechanism of STAT3 hypomethylation mediated by TET2 involved in benzene hematopoietic toxicity and to verify that TET2 may be a new potential target for early screening in population exposed to benzene.

苯造血毒性防治重点在于阐明机制和寻找早期标志物。课题组前期研究发现STAT3低甲基化参与调控苯的造血毒性，但机制不清。TET2作为DNA甲基化的重要调节因子，是调控造血的重要蛋白，提示TET2可能是苯造血毒性的关键分子，通过介导STAT3低甲基化调控造血干/祖细胞的更新分化，继而导致苯的造血毒性。近期课题组成功建立了人源造血干/祖细胞的培养体系，本项目拟以此为基础利用高内涵、PCR荧光探针、测序、甲基化PCR、RNAi等方法研究苯代谢物1,4-苯醌致造血干/祖细胞更新分化异常中TET2表达、STAT3甲基化的变化规律及TET2介导STAT3低甲基化在造血干/祖细胞更新分化异常中的作用；在已建立的苯职业人群队列中分析苯暴露，TET2、STAT3表达和血象之间的相关性及量效关系。阐明TET2调控STAT3低甲基化在苯造血毒性中的作用和机制，探索将TET2用于苯接触人群造血危害早期筛查的新靶点。

苯造血毒性防治重点在于阐明机制和寻找早期标志物。TET2作为DNA甲基化的重要调节因子，是调控造血的重要蛋白，提示TET2可能是苯造血毒性的关键分子，但TET2在苯致造血毒性中的作用和机制不清。本课题从人群及细胞两个层面，揭示TET2在苯暴露人群和苯代谢物染毒细胞中表达情况及其在苯毒性中的作用；阐明TET2介导苯血液毒性的机制。在苯暴露职业人群中检测TET（TET1、TET2和TET3）基因和蛋白的表达，发现低剂量苯暴露人群全血细胞和血清中TET2基因和蛋白表达，与对照人群相比均显著升高(p<0.05)，苯暴露组TET2的表达与淋巴细胞计数（LYPMH）存在负相关关系(p<0.05)，与尿苯代谢物苯巯基尿酸水平呈现正相关关系(p<0.05)。体外实验研究发现苯代谢物1,4-BQ暴露导致TET2基因和蛋白表达升高，且具有剂量依赖性(p<0.05)。提示TET2在苯血液毒性中发挥着重要作用，是苯血液毒性发生的关键分子。人群结果显示低剂量苯暴露导致焦亡经典信号通路基因Caspase1和IL-1β表达升高，伴随血清中IL-1β释放增加（p<0.05），衰老执行蛋白p53表达增加而G1期调控蛋白Cyclin D1表达降低（p<0.05），提示苯暴露可能通过细胞焦亡诱发炎症衰老导致血液毒性。体外采用慢病毒和小分子抑制剂干扰TET2表达后，1,4-BQ 染毒所致的细胞焦亡（NLRP3、Aim2、Caspase1和IL-1β）及毒性效应分子（IL-8，IL-6，p53和p21）及ATF3启动子区羟甲基化水平和基因蛋白表达均显著下降，表明TET2通过靶向调控ATF3表达促进细胞焦亡，继而导致苯的毒性效应。本项目阐明了TET2在苯血液毒性中的重要作用及TET2通过调控ATF3诱导细胞焦亡参与苯血液毒性的机制，TET2可作为苯暴露人群早期危害筛查和健康监护的潜在分子。