以miR-363为重要节点的miRNA网络异常在Graves病Treg细胞功能失调中的作用及机制

the dysfunction of regulatory T cells (Treg) is one of the most important reasons of Graves' disease (GD) development. However, to our knowledge, the study of the effects of miRNA on the function of Treg in GD development is still unreported. Our preliminary study showed that the function of Treg cells in GD patients were significantly down-regulated and the expressions of many miRNA were changed. According to the above-mentioned results, we speculate that the node miRNA-miR-363, which is significantly upregulated, can inhibit the activity of Notch and STAT signaling pathway and down-regulate the expression of key genes related to the function of Treg cells by inhibiting its target genes Notch1, MAML3 and STAT4. The abnormality of the regulation networks of miRNA interfere with the normal function of Treg cells and promote the production of TSAb resulting in the development of GD. To test this hypothesis, we plan to study the mechanisms of the up-regulation of miR-363 expression in Treg cells of GD patients and the mechanisms of the inhibition to Treg cells by reporter gene vector and proteomic technologies. We will also observe the effects of miR-363 on the mouse model of GD. Finally, we hope to reveal the mechanisms of the effects of miR-363 on the dysfunction of Treg cells and the development of GD. This proposal will help us to understand the effect mechanisms of miRNA on Treg cells and the development of GD and other autoimmune diseases and guide the design of miRNA drugs for immunotherapy.

调节性T细胞(Treg)异常是Graves病(GD)的重要病因，但miRNA作为关键免疫调控分子在这些异常中的作用和机制目前仍未见研究报道。我们前期研究发现初发未治疗GD患者外周血Treg细胞功能明显下降，多个miRNA显著调变；数据分析提示显著上调的重要节点miR-363能通过其靶基因Notch1、MAML3和STAT4抑制Notch和STAT通路，下调Treg细胞关键基因表达，这种调控网络异常抑制了Treg细胞功能，促进TSAb产生而发生GD。为验证这一假说，本申请将用报告基因和蛋白组等技术，研究miR-363在GD患者Treg细胞中上调及抑制Treg细胞功能的机制，观察miR-363对GD小鼠病情的影响，揭示miR-363在GD发病和Treg细胞异常中的作用机制。本项目将有助于理解miRNA对Treg细胞的调控以及对GD等自身免疫疾病的作用和机制，并指导miRNA免疫治疗药物的设计.

Graves＇disease（GD）是一种常见的器官特异性自身免疫疾病，是自身免疫性甲状腺病(AITD) 的主要类型之一。GD的主要病理特征是存在甲状腺组织特异的自身抗体（如TRAb），这是GD的主要病因和标志物。GD的发病机制复杂，遗传和感染等众多因素都起着重要作用，但这些内外因素如何破坏TSHR的自身免疫耐受而产生TRAb的确切分子机制迄今仍未完全阐明。调节性T细胞(Treg)是免疫调控的关键细胞，在维持机体正常免疫稳态和自身免疫耐受中发挥关键作用，在多种自身免疫疾病中都存在显著的Treg细胞异常。miRNA是一类长约18-25nt的具有调控功能的非编码小分子RNA，在Treg细胞分化、增殖与功能中都发挥重要作用，是很多自身免疫疾病发生和进展的关键因素之一。此外，一个miRNA能调控多个靶基因，影响多条信号通路，形成以该miRNA为节点的调控网络，通过网络间的协调作用产生最终的调节效应，这使miRNA的作用具有整体、协调和复杂的特点。我们的研究表明GD患者外周血Treg细胞的数量和功能都明显下降，而miR-363表达则显著升高。CD4+T细胞转染miR363后Notch1、MAML3和STAT4的表达显著下降，同时STAT4的活性形式，即Tyr693磷酸化的Stat4以及活化的Notch1也都显著下调。荧光素酶报告载体的结果进一步证明调控Treg细胞关键功能分子IL10和FoxP3表达的Notch1和STAT4是miR363的靶基因。这些结果表明在多种遗传和环境因素作用下，GD患者外周Treg细胞miR-363上调，从而使其靶基因Notch1和STAT4下调，导致Notch和STAT信号通路抑制；这两条信号通路的抑制效应叠加，使FOXP3和IL-10的表达显著下降，Treg细胞功能受损、数量下降，最终促进了TSHR免疫耐受破坏。本课题揭示了miR-363在GD患者Treg细胞异常中的作用机制，有助于理解miRNA对Treg细胞的调控以及对GD等自身免疫疾病的作用和机制，并指导miRNA免疫治疗药物的设计.