DEHP与TCDD共暴露与乳腺癌发生发展的受体交互应答机制研究

As prototypical Endocrine Disruptors (EDCs), both Di-(2-ethylhexyl)phthalate (DEHP) and 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) could produce a diverse spectrum of biological and toxic effects in a wide range of species and tissues, through binding Estrogen Receptor (ER) and Aryl hydrocarbon receptor (AhR) directly or indirectly. Breast cancer is one of endocrine-related tumors and is ranked the leading cancer in Chinese women. Cumulative exposure of EDCs has been suspected as the most potential environmental risk factor for breast cancer. Based on our previous studies, we intend to conduct studies with DEHP and TCDD to elucidate mechanisms of the cross-talk of ER and AhR with the goal of providing a framework for assessing cumulative effects of EDCs on the development of mammary gland tumor. We will establish models of mammary gland epithelial cells, breast cancer cell lines and human breast cancer cells in tumor-bearing nude mice with the exposure of DEHP and TCDD to investigate the effects of malignant transformation and tumor growth for breast cancer. By methods of cell transfection and gene knockout of AhR/ARNT, XRE, ER and ERE in cells and animals, we expect to illustrate different hypothetical mechanisms of ER-AhR crosstalk under the sum / synergistic / antagonistic effects of DEHP and TCDD: (A) by competing for common cofactors; (B) by competing for promoter binding leading to inhibition of transcription. (C) regulate the levels of circulating E2 by controlling the gene expression and epigenetic changes of CYPs. Overall, we focus on the mechanisms of EDCs affecting tumor development at the level of receptors, providing new clues and basis for a comprehensive understanding of the environment, gene and tumor interactions.

DEHP与TCDD作为典型的内分泌干扰物(EDCs)，可与雌激素受体(ER)和芳香烃受体(AhR)结合诱导产生多种毒性效应。乳腺癌作为内分泌相关肿瘤，EDCs的混合暴露被认为是其发生发展的主要环境因素之一。在前期研究基础上，本项目拟从ER与AhR交互应答的机制出发，探索二元暴露致乳腺癌发生发展的联合效应。拟建立DEHP和TCDD暴露促进正常乳腺上皮细胞恶性转化和癌细胞侵袭转移的细胞模型以及人乳腺癌细胞荷瘤裸鼠模型，以AhR/ARNT，XRE，ER和ERE基因操纵方法在体外或体内水平探索分析DEHP与TCDD共暴露对人乳腺癌发生发展所产生的效应，通过细胞辅活化子的竞争性结合，受体与靶基因启动子区域募集反应机制，及E2合成代谢及关键基因表观遗传改变等方面阐明ER-AhR交互应答在乳腺癌发生发展中的作用，期望明确EDCs共暴露在受体层面的机制，为全面理解肿瘤-环境-基因间的关联提供理论依据。

本研究旨在探讨邻苯二甲酸-单-2-乙基己酯（MEHP）暴露、四氯二苯并-对-二噁英（TCDD）暴露、MEHP与TCDD共暴露对乳腺癌细胞MCF-7的增殖、迁移和侵袭，以及对芳香烃受体（AhR）信号通路的影响，以阐明MEHP与TCDD共暴露在乳腺癌发展过程中的作用及机制。.1. MEHP暴露6h和48h均可抑制细胞增殖，具体表现为MEHP 10μM、20μM和40μM暴露6h后可显著抑制细胞增殖，MEHP 1μM、10μM和20μM暴露48h可显著抑制细胞增殖，其他浓度暴露不影响细胞增殖；转染siRNA-AhR后并不能缓解MEHP对细胞增殖的抑制作用，表明MEHP抑制增殖作用不依赖AhR；2. TCDD单独暴露不影响细胞增殖。MEHP与TCDD共暴露6h和48h对细胞增殖的影响存在交互作用，而共暴露12h和24h对细胞增殖无影响。MEHP与TCDD共暴露后，细胞增殖能力显著降低，表明MEHP与TCDD共暴露可抑制细胞增殖；AhR干扰后并未缓解MEHP与TCDD共暴露对细胞增殖的抑制作用，表明共暴露对细胞增殖的影响不依赖AhR；3. MEHP暴露可促进细胞迁移和侵袭，表现为MEHP 1μM、10μM和100μM暴露24h均可显著促进细胞迁移，MEHP 10μM和100μM暴露48h后细胞迁移能力显著高于对照组，MEHP 10μM和100μM暴露48h后细胞侵袭能力显著高于MEHP 1μM；转染siRNA-AhR后MEHP 100μM诱导的细胞迁移作用减弱，但是转染siRNA-AhR不影响MEHP 100μM介导的细胞侵袭。4. PCR-Array结果表明，MEHP暴露可显著上调23个基因表达，下调8个基因表达；TCDD暴露可显著上调22个基因表达，下调6个基因表达。在18个基因表达上，MEHP可产生与TCDD相同的作用，包括14个上调的基因和4个下调的基因。5. MEHP与TCDD共暴露对AhR和CYP1A1基因表达存在拮抗性交互作用，MEHP介导的AhR基因表达被TCDD抑制，同时TCDD介导的CYP1A1基因表达可被MEHP抑制；MEHP与TCDD共暴露对CYP1B1的表达交互作用无统计学意义；转染siRNA-AhR后，TCDD诱导的CYP1A1显著降低，且MEHP对TCDD介导的CYP1A1表达的拮抗作用消失。