Lnc-DSG1-6调控MDM2介导宫内高雌暴露致子代甲状腺功能紊乱的作用和机制研究

The effect of intrauterine adverse environment on the health of offspring is the hot issue concerned by the current research. Our preliminary epidemiological survey found that the intrauterine high estrogen environment in early pregnancy can induce thyroid dysfunction of offspring, but the mechanism is unclear. Further microarray analysis of long non-coding RNAs revealed that high estrogen up-regulated the expression of lnc-DSG1-6 and murine double minute 2 (MDM2), which plays a key role in the thyroid signaling pathway. Lnc-DSG1-6 combined to MDM2 directly and the expression of lnc-DSG1-6 was closely correlated to MDM2. In this light, we proposed the hypothesis that "high estrogen exposure during pregnancy leads to thyroid dysfunction via lnc-DSG1-6-MDM2". This project intends to identify the key role of lnc-DSG1-6 and MDM2 in thyroid dysfunction induced by high estrogen exposure via interference or overexpression of lnc-DSG1-6 or MDM2, to elucidate the regulatory model of lnc-DSG1-6 and MDM2 by RIP, to explore the regulation molecular mechanism of high estrogen on lnc-DSG1-6 transcription by luciferase reporter system, to validate the role of lnc-DSG1-6 in thyroid function through mouse models of lnc-DSG1-6 interference or overexpression, to confirm the correlation between maternal high estrogen level and thyroid lnc-DSG1-6 expression by mouse model of intrauterine high estrogen, and to transfer the results of research to clinical medicine. This may do a great favor in preventing embryo-fetal origin of diseases caused by adverse maternal environment.

宫内不良环境对子代健康的影响日益受到关注。我们前期研究已证实宫内高雌环境可诱发子代甲状腺功能紊乱，机制不详。进一步长链非编码RNAs芯片结果发现高雌致lnc-DSG1-6及甲状腺通路重要基因MDM2表达上调，lnc-DSG1-6与MDM2直接结合且表达高度相关。据此，我们提出“早孕期高雌激素暴露通过lnc-DSG1-6-MDM2诱发子代甲状腺功能紊乱”这一假说。本项目拟在甲状腺细胞中敲减或过表达lnc-DSG1-6和MDM2以明确两者在高雌干扰甲状腺激素合成中的关键作用，借助RIP等技术阐明两者调控模式，荧光素酶报告系统分析高雌调控lnc-DSG1-6的分子机制；lnc-DSG1-6干扰及过表达小鼠证实其在甲状腺代谢中作用；高雌小鼠模型验证子代甲状腺组织lnc-DSG1-6水平与孕鼠高雌相关性；并行临床研究探讨lnc-DSG1-6作为预警指标可行性。为孕期不良环境致胎源性疾病研究开拓思路。

研究背景：本课题组前期研究发现宫内高雌环境可诱发子代甲状腺功能紊乱，高雌致lnc-DSG1-6及甲状腺通路重要基因MDM2表达上调，据此我们提出“早孕期高雌激素暴露通过lnc-DSG1-6-MDM2诱发子代甲状腺功能紊乱”这一假说。本项目拟明确lnc-DSG1-6与MDM2在高雌干扰甲状腺激素合成中的作用，阐明两者调控模式，并通过小鼠模型验证其在甲状腺代谢中作用。.研究内容：明确Nthy ori3-1细胞中lnc-DSG1-6敲减和过表达对甲状腺激素分泌的影响； lnc-DSG1-6干扰及过表达小鼠的构建及甲状腺功能检测；Nthy ori3-1细胞中lnc-DSG1-6或MDM2表达异常对对方表达的影响；lnc-DSG1-6与MDM2蛋白相互关系；MDM2敲减和过表达对甲状腺激素合成的影响；MDM2表达异常对甲状腺功能途径中关键酶的影响。.研究结果：雌激素处理Nthy ori3-1可上调lnc-DSG1-6水平，提高MDM2表达，调控甲状腺功能代谢，干扰lnc-DSG1-6可以下调Mdm2表达并抑制甲状腺滤泡细胞的增殖。Nthy ori3-1细胞siRNA干扰lnc-DSG1-6表达后T4表达显著下降，过表达lnc-DSG1-6后T4表达显著上调。进一步小鼠动物模型体内验证， lnc-DSG1-6干扰小鼠模型中T4水平显著下降，lnc-DSG1-6过表达模型中T4水平显著增加。MDM2干扰后lnc-DSG1-6表达无显著改变，lnc-DSG1-6干扰后MDM2表达显著下降。RNA免疫共沉淀实验MDM2蛋白跟lnc-DSG1-6紧密结合。Nthy ori3-1细胞中MDM2干扰后，T4表达显著下降，过表达MDM2后T4表达显著上调。siRNA干扰MDM2表达后TPO表达显著下降。提示MDM2主要通过TPO发挥作用。分析了lnc-DSG1-6启动子区的ER结合位点，并进行了进一步染色质免疫共沉淀（ChIP）验证候选DNA片段与ER的结合能力，通过构建荧光报告素酶质粒、DNA定点突变确认该ERE并不具有明显的转录调控活性。.结论：在甲状腺滤泡细胞和小鼠中干扰和过表达lnc-DSG1-6后甲状腺T4水平相应地降低和增高，从体外和体内两方面证明lnc-DSG1-6调控甲状腺功能。lnc-DSG1-6直接结合并调控MDM2表达，MDM2通过TPO调控甲状腺激素水平。