Foxe1介导宫内高雌环境影响子代甲状腺功能的机制研究

The safety of the children conceived through assisted reproductive technology (ART) has attracted much attention recently. The supra-physiological state of high estrogen induced by ovulation stimulation might impair the thyroid function of the children, but its mechanism is still unclear. Our previous study found that maternal high estrogen environment might lead to thyroid dysfunction and abnormal expression of transcription factor Foxe1 in offspring, and in the present study we try to explore the underlying mechanism by which estrogen influences thyroid function via Foxe1. Mouse model of intrauterine high estrogen environment is used to clarify the expression and location of Foxe1 in different stages of thyroid development of offspring; mRNA-Seq screening is adopted to correlate thyroid function parameters and the variation of corresponding key molecules of thyroid hormone synthesis and metabolism; an over-expression system is used to clarify the impact on the expression of downstream key target genes of Foxe1; in vivo and in vitro models are used to detect the Foxe1 promoter methylation status and expression of DNA methyltransferases (Dnmts) after exposure to high estradiol environment; further experiments are performed to determine whether estrogen regulates Dnmts through estrogen response element (ERE); specific gene knockout technology CRISPR/Cas9 is performed in cell culture, to correct the abnormal key enzymes of methylation regulation, and to analyze whether correction of abnormal methylation modification can reduce the adverse effect. The study tries to uncover the mechanism of abnormal thyroid function induced by maternal high estrogen environment, which may provide innovation and theoretical basis for studying embryo-fetal origin of diseases.

辅助生殖技术出生子代的安全性日益成为关注焦点。已证实促排卵使母体处于非生理性高雌激素状态，影响子代甲状腺功能，但机制不详。本课题组在前期发现宫内高雌致子代甲状腺功能异常及Foxe1表达改变的基础上，将深入研究Foxe1介导的宫内高雌对子代甲状腺功能的影响机制。拟建立宫内高雌小鼠模型，明确不同发育阶段子代甲状腺Foxe1的定位及表达；利用mRNA-Seq技术，比较甲状腺激素合成代谢关键因子及相应甲状腺功能指标改变；应用过表达体系阐明Foxe1对下游关键靶基因表达影响；利用在体和体外模型检测高雌作用下Foxe1启动子区甲基化水平及甲基转移酶的水平，验证雌激素是否通过ER反应元件调控Dnmts；通过CRISPR/Cas9特异性基因敲除技术，纠正异常甲基化调控关键酶，分析改善甲基化修饰异常后对不良影响的消减作用。揭示宫内高雌环境致子代甲状腺功能异常的可能机制，为其他发育源性疾病研究开拓思路。

高雌组子代小鼠在3周和8周时甲状腺激素分泌明显异常，高雌组子代在妊娠第18天、出生后3周和8周的Foxe1表达显著上调。甲基化实验证实高雌组子代在不同发育阶段的Foxe1基因启动子区域CpG岛的甲基化水平均显著低于对照组，同时也检测到高雌组Dnmt3a表达显著降低。甲状腺细胞原代培养中加入不同浓度雌激素，高雌组Foxe1启动子区甲基化水平显著性降低。另外，荧光素酶报告实验说明雌激素可能并不是通过其经典核受体途径来调控Dnmt3a表达。在甲状腺滤泡细胞系中雌激素能够浓度依赖性地促进甲状腺滤泡细胞的增殖，下调Dnmt3a和上调甲状腺转录因子Foxe1的表达，而加入雌激素受体阻断剂ICI182780后，能显著阻断雌激素的调控作用。. 在string数据库中，FOXE1也与其他甲状腺转录因子、甲状腺激素合成基因以及甲状腺激素运输基因密切相关。甲状腺滤泡细胞系过表达FOXE1后TPO的表达显著性上升，免疫组化实验也证实高雌组子代TPO蛋白表达显著性增高。对子代甲状腺组织的表达谱芯片测序结果提示，高雌组子代甲状腺激素合成、代谢及分子调控网络的很多相关基因出现了异常。lncRNAs芯片分析发现高雌激素处理会导致人甲状腺滤泡细胞系lncRNAs表达谱发生改变。KEGG分析和上游通路分析发现有15个差异基因受雌激素调控，其中包括与甲状腺代谢密切相关的mdm2基因的表达异常上调。. 综上所述，体内外实验证实宫内高雌激素环境暴露可引起子代成年后甲状腺功能紊乱，其中甲状腺组织中FOXE1表达的增加起了关键作用，进一步芯片实验提示MDM2和lncRNA可能共同参与甲状腺代谢的调控。高雌环境可能通过调控启动子区甲基化减少，进而上调FOXE1的表达，从而介导甲状腺代谢的干扰。本项目的开展，为辅助生殖技术中的异常甾体激素环境影响子代安全性的表观遗传机制提供了科学依据。