DNA羟化酶TET1抑制甲状腺功能的机制研究

Thyroid hormone (TH) plays a critical role in maintaining basal metabolic rate and the development of target organs, including central nervous system, skeletal and reproductive systems. Any abnormality in the intricate system of thyroid gland and hormone synthesis can introduce thyroid dysfunction and bring systematic damages even irreversible disabilities. Thyroid function is generally regulated by thyrotropin receptor (TSHR) and its downstream transcritpion factors including TTF1/2, PAX8, and CREB. However, the importance of epigenetic mechanisms in these key processes remains largely unknown. TET1 is a DNA hydroxylase that convert 5-methycytosine (5-mC) into 5-hydroxylmethylcytosine (5-hmC), and is involved in regulating gene expression through DNA demethylation and chromatin remodeling. Our preliminary data showed that severe hypothyroidism was observed in the thyroid glands of transgenic mice with thyroid-specific knock-in of BrafV600E (TPO-Cre/LSL-BrafV600E), resulting in loss of body weight and fertility in mice. Interestingly, we found that mice restored thyroid function and reproductive ability when Tet1 was knocked-out specifically in the thyroid gland of TPO-Cre/LSL-BrafV600E mice. These data suggest that TET1 negatively regulates thyroid function. The aims of this project were to identify the target genes of TET1 and explore the mechanism underlying the inhibitory effect of TET1 on thyroid function by using RNA-Seq, ChIP-Seq and cell bioenergy analysis. This study will help us to better understand the role of epigenetic mechanisms in regulating thyroid function and provide therapeutic startegies for thyroid dysfunction.

正常的甲状腺激素（TH）水平是维持机体基础代谢、多器官发育及生理功能的关键。甲状腺功能异常是不容忽视的内分泌疾病，可累积全身多个系统，近年来发病率急剧增加，严重影响人们的身心健康。TH合成及分泌主要依赖甲状腺刺激激素受体（TSHR）及下游转录因子的调控，而表观遗传学调控机制目前尚无报道。DNA羟化酶TET1介导DNA主动去甲基化及染色质重塑，参与基因表达调控。我们的前期研究发现在甲状腺中引入BrafV600E突变可导致小鼠发生严重的甲状腺功能减退，使之丧失生殖能力，而联合敲除Tet1基因可有效恢复小鼠的甲状腺功能及其生育能力，提示TET1可能负向调控甲状腺功能。本项目旨在此基础上拟通过RNA-Seq、ChIP-Seq、细胞生物能量分析等技术手段筛选TET1的下游靶基因，阐明其抑制甲状腺功能的分子机制，从表观遗传学视角完善TH合成及分泌的调控机制，为甲状腺功能异常提供新的治疗策略和思路。

甲状腺疾病发病率逐年上升，异常的甲状腺激素水平累积全身多器官，引起生长发育和机能异常。DNA羟化酶TET1能够结合于基因组的CpG岛，介导DNA去甲基化。此外，TET1在染色质重塑过程中也扮演重要角色。目前TET1与疾病的关联多集中在肿瘤和免疫治疗等领域。有证据表明，甲状腺特异引入BrafV600E突变可诱导小鼠发生乳头状甲状腺癌；同时，由于BrafV600E突变破坏甲状腺滤泡上皮细胞的生理功能，导致小鼠发生严重的甲状腺功能低下（甲减），进而使其丧失生育能力。我们在前期体内实验中发现在甲状腺中特异性敲除Tet1基因后可恢复BrafV600E突变小鼠的体重和游离T3（FT3）的水平，而甲状腺激素水平上升使得小鼠再获生殖能力。这些结果提示我们，TET1可能负向调控甲状腺功能。接下来，我们构建单纯甲状腺敲除Tet1的转基因小鼠。结果发现，与野生小鼠相比，敲除Tet1的小鼠甲状腺激素水平上升，并伴随体重减轻、心率、血压增高及肛温较高等全身高代谢的症状。同时，RNA-Seq的结果显示，特异性敲除Tet1的小鼠甲状腺组织中转录因子Pax8基因的mRNA水平上调。此外，我们利用免疫组化实验证实Pax8及下游靶分子Nis和Tpo的蛋白表达水平均显著上调。接下来，我们探索TET1负向调节PAX8的分子机制。我们利用免疫共沉淀（Co-IP）和ChIP-qPCR等实验证实TET1与去乙酰化酶HDAC1存在相互作用，招募后者到PAX8的启动子区，降低其H3K27Ac乙酰化水平下降，进而抑制其转录。当敲减TET1后，HDAC1对PAX8的转录抑制作用被解除，进而促进甲状腺激素的合成。同时，我们发现，相较正常的甲状腺组织，TET1在甲状腺功能亢进（甲亢）患者的组织标本中表达下调。此外，我们发现在部分甲亢患者血清外泌体中miR-29c-3p的水平显著升高，而双荧光素酶报告系统证实miR-29c-3p可靶向TET1 mRNA的3’ UTR区，抑制其翻译。综上，我们的实验结果提示甲亢患者血清外泌体中升高的miR-29c-3p通过靶向TET1解除其对PAX8的转录抑制作用，进而促进甲状腺激素合成。本研究揭示了一个全新的甲亢发生的分子机制，为甲亢的治疗提供新的思路和策略。在该项目的支持下，申请人以通讯作者发表SCI论文6篇。