EZH2介导二噁英影响ER阳性乳腺癌发生发展的表观遗传机制研究

Dioxins are typical persistent organic pollutants (POPs) and endocrine disruptor chemicals (EDCs), which can affect the development of breast cancer by epigenetic modifications. As a histone methyltransferase enzyme, EZH2 catalyzes histone methylation, mediating silencing of target genes. Our previous study found that the aryl hydrocarbon receptor (AhR) and estrogen receptor (ER) presents cross-talk effects after the exposure of dioxin in breast cancer cells. It is hypothesised that the effects of dioxin are mediated by EZH2 through epigenetic modifications during the development of ER-positive breast cancer. Normal mammary epithelial cells, breast cancer cells and the population samples in typical area will be adopted in the present study. Cells will be transfected with EZH2 overexpression or EZH2-siRNA Vector. DNA methylation and histone methylation / acetylation of target genes will be detected by Chromatin Immunoprecipitation (ChIP). The protein interactions of receptors, EZH2, co-activators and breast cancer-related proteins (BRAC1 etc.) will be tested by Co-Immunoprecipitation (Co-IP). We also analyze the combination and reaction of AhR, ER, co-activators, BRAC1 with EZH2 promoter region. Samples of human breast tissues will be collected and the concentrations of dioxins will be evaluated. The mechanisms mediated by EZH2 are verified in human breast tissues, which will be used to the analysis of EZH2 expression and epigenetic changes of associated genes. The results of this project will provide scientific basis for researches of epigenetic mechanisms for environmental factors and cancer.

二噁英是典型的持久性有机污染物和内分泌干扰物，可通过表观遗传改变影响乳腺癌的发生发展。EZH2为一种组蛋白甲基转移酶，催化组蛋白甲基化，影响表观遗传修饰，介导靶基因沉默。课题组前期研究发现二噁英作用于乳腺癌细胞，芳香烃受体(AhR)与雌激素受体(ER)呈现交互应答效应。我们假设EZH2通过表观修饰介导二噁英影响ER阳性乳腺癌的发生发展。本项目拟采用正常乳腺上皮细胞、乳腺癌细胞、典型区域的人体生物样本机制验证三种方式进行。在细胞模型中进行EZH2的干扰和过表达质粒转染，检测受体，EZH2，辅活化因子，乳腺癌相关基因(BRAC1等)的DNA甲基化，组蛋白甲基化和乙酰化，蛋白间的相互作用，及在EZH2启动子区域的募集反应。通过人组织样本进行机制验证，测定乳腺组织中二噁英类物质水平，同时分析EZH2相关基因的表达和表观遗传学改变。对假设进行验证，为“环境因素—表观遗传学—肿瘤”的研究提供科学依据。

二噁英是典型的持久性有机污染物和内分泌干扰物，属于IARC确定的一类致癌物，可通过介导表观遗传改变从而影响乳腺癌的发生发展。EZH2为一种组蛋白甲基转移酶，其可作为PRC2（多梳蛋白复合物2）的催化亚基发挥功能，催化组蛋白甲基化，影响表观遗传修饰，介导靶基因沉默，也可经由其自身胞内定位的改变而影响乳腺癌的增殖和迁移。因此，我们对EZH2在TCDD影响乳腺癌的发生发展中的作用进行了深入研究。.本项目采用正常乳腺上皮细胞、乳腺癌细胞、乳腺癌人体生物样本三种方式进行假说验证。在MCF7、MCF-10A细胞中进行TCDD染毒暴露后，通过CCK-8、细胞周期检测其对增殖的影响，结果显示TCDD促进了细胞增殖。通过划痕、Transwell实验检测其对细胞迁移的影响，发现TCDD对两株细胞迁移并无明显影响。并通过q-PCR，Western Blotting等实验对乳腺癌相关基因(BRCA1，BRCA2等），芳香烃受体及其下游基因，EZH2及组蛋白甲基化表达水平进行检测，结果显示TCDD暴露后激活了两株细胞的AhR信号通路，及显著上调了EZH2、MYC等与乳腺癌发生发展有关的基因的表达，但EZH2并非为AhR下游靶基因。并通过对EZH2基因敲除后进行测序分析，结果显示，ACKR3为EZH2基因的典型靶基因，其与细胞的迁移密切相关，后续还应对其继续深入研究。通过测定人乳腺癌组织样本二噁英类物质水平，及结合乳腺癌患者临床病理分析，发现乳腺癌的发生可能与PCDF的暴露水平相关，且癌旁脂肪组织可能成为日后研究二噁英内暴露的重要生物样本。这可以为二噁英与乳腺癌的机制探索提供数据支持，为“环境因素—表观遗传学—恶性肿瘤”的研究提供科学依据。