Tet介导的DNA去甲基化在苯致基因组不稳定及白血病中的作用

Global Genomic hypomethylation and related genetic instability are both hallmarkers of cancer cell. Benzene could induce global hypomethylation and DNA damage in vitro and in vivo. While the underlying mechanisms and their potential roles in benzene-induced leukemia keep unclear. In 2011 active DNA demethylation in mammals was confirmed. Ten-eleven translocation (Tet) protein family members oxidize 5-methyl-Cytosine (5mC) stepwise via 5-hydroxymethyl- cytosine (5hmC) and 5-formyl-cytosine (5fC) to 5-carboxy-cytosine (5caC) in an enzymatic activity-dependent manner. Tet mutations are frequently involved in human myeloid malignancies and often affect the catalytic activity. In addition, the pilot experiments in our Lab showed that benzene increased Tet mRNA level in vitro. Accordingly, we hypothesize that Tet-mediated DNA demethylation plays an important role in benzene-induced genetic instability and leukemia. The research programme includes three sections. In in vitro cell model, the effects of benzene and its metabolites hydroquinone and benzoquinone on Tet expression, DNA methylation level, percentage of 5hmC, 5fC and 5caC, loss of imprinting of H19 and DNA damage will be studied. Whether the toxic effects of benzene are affected by up- or down-regulation of Tet is also to be tested. In order to elucidate how benzene affects Tet, the expression profiles of miR29a/29b, which is reported to be related to global hypomethylation and one of the predicted target genes of which is Tet, will be observed when the cells are treated by benzene. The direct evidence for the interaction of miR29a/29b and Tet will be obtained by 3′-UTR of Tet vector construction and luciferase reporter assay. The method for 5hmC, 5fC and 5caC detection is liquid chromatography and mass spectrometry (LC-MS) and thin layer chromatography (TLC). Bisulfite-sequencing, immunohistochemistry assay, miceller electrokinetic capillary chromatography and pyrosequencing are used to detect global methylation levels. The main results of the in vitro tests will be validated in mouse bone marrow cells and peripheral lymphocytes during or after a short-term (2 weeks) or a long-term (64 weeks) benzene exposure by inhalation. Benzene-induced hematotoxicology will be focused on in animal test. In occupational epidemiology study a total of 420 healthy subjects and benzene poisoning patients with different benzene exposure levels will be recruited. Individual exposures are assessed using personal air monitoring and urinary concentrations of benzene, t,t-MA and S-PMA. The relationship between air benzene exposure, individual exposure and global hypomethylation in peripheral lymphocytes are analyzed. The proposed study will shed light on the mode of action for benzene hematotoxicity and carcinogenesis, and the feasibility of global hypomethylation as an biomarker for benzene exposure.

基因组低甲基化导致基因组不稳定（GI）是肿瘤细胞重要特征，血液系统致癌物苯可导致基因组低甲基化和GI，但机制不明。2011年，Tet酶催化5mC经由5hmC、5fC和5caC而主动去甲基化过程得以证实，且Tet在白血病中存在广泛突变，预实验也显示苯干扰Tet表达，因此"苯靶向Tet导致基因组低甲基化是苯致白血病的关键事件"成为亟待验证的化学致癌机制假说。本课题通过细胞试验观察苯对Tet表达、DNA甲基化、稀有碱基含量、印记基因表达和DNA损伤的影响，探讨Tet表达改变对苯毒性的影响及苯通过miR29a/29b调控Tet的机制；通过短期和长期动物试验，在靶细胞上验证体外试验的结果并将观察终点推进到血液毒性；通过收集不同苯暴露水平的健康人和苯中毒患者，分析上述指标与空气苯、体内代谢产物及中毒程度的关系，探讨基因组低甲基化作为苯暴露生物标志物的可行性。本研究对于完善苯毒作用模式具有重要科学意义。

为了探讨环境污染物暴露与基因组甲基化水平的关系、调控机制及基因组甲基化水平作为生物标志物的应用价值，本项目从体外细胞培养、动物实验及人群研究三个层面，观察了镉（CdCl2）、全氟辛烷磺酸（PFOS）、苯（Benzene）、双酚A（BPA）对基因组及单个基因甲基化水平的影响；初步探讨了Tet酶改变（镉）、线粒体毒性（PFOS）与DNA甲基化的关系；利用光照改变建立生物节律改变的小鼠模型，探讨生物节律改变与苯、BPA对小鼠肝脏代谢、血液毒性及遗传损伤的交互效应；在石化工人中观察了低剂量苯暴露与遗传损伤及MGMT基因甲基化的关系；对孕期妇女职业史、生活作息制度的调查，观察孕期生物节律紊乱对出生结局的影响，以及孕期生物节律紊乱、环境污染暴露对出生时基因组甲基化及出生端粒长度的影响。研究发现：在CdCl2短期暴露下，随着染毒剂量和染毒时间的增加，Tet酶mRNA和蛋白表达水平呈逐渐升高的趋势，且全基因组DNA也表现出去甲基化改变；镉作用于人胚肾细胞HEK293时，甲基化酶在其全基因组甲基化水平改变方面可能发挥了一定的调控作用；CdCl2亚急性染毒可以诱导小鼠肝肾功能损伤，且对全基因组甲基化水平和去甲基化酶Tet1表达呈现出性别差异影响，雄性小鼠的肝肾组织Tet1表达参与调控全基因组DNA甲基化水平改变；模拟轮班作业的光照改变干扰小鼠的生物节律，并且与苯或BPA在小鼠肝脏代谢、血液毒性及遗传损伤的一些参数方面方面存在交互效应；与对照组相比，长期低剂量（低于接触限制）苯接触工人DNA损伤（彗星实验，尾部DNA含量，%）的差异具有统计学意义（6.51±2.03 vs. 5.84±2.24，P=0.012），MGMT基因启动子区呈现总体及位点特异的低甲基化，9个位点的平均甲基化水平（%5mC）为3.21±0.83 vs. 3.59±0.90，P=0.010。此外，MGMT基因甲基化水平与尿液中SPMA含量呈现负相关，提示MGMT基因低甲基化可以作为低剂量苯暴露及DNA损伤的潜在生物标志物；孕期生物节律改变可能与不良出生相关，相关机制有待深入研究；meta分析显示基因组低甲基化是肿瘤不良预后的生物标志物。