E3泛素连接酶HUWE1在免疫性血小板减少性紫癜Treg/Th17免疫失衡中的作用及机制研究

Immune thrombocytopenia (ITP) is an organ-specific autoimmune disease, characterized with decreased platelet count and increased bleeding risk. It is well known that Treg/Th17 imbalance plays an important role in ITP development. However, the molecular mechanism of Treg/Th17 imbalance in ITP is unclear. Our preliminary results showed that ubiquitin E3 ligases HUWE1 was significantly increased in CD4+T cells from ITP； Downregulation of HUWE1 could partially.improved Treg/Th17 balance in ITP； Further study showed that HUWE1 could directly interact with Ets-1 and mediated Ets-1 ubiquitylation and degradation. In.addition, HUWE1 was the target of miR-125a-5p in CD4+T cells. As a result, we speculated that downregulation of miR-125a-5p contributed to the increase of HUWE1 expression, which resulted in the decrease of Ets-1expression in CD4+T cells from ITP..The project intends to study 1）the role of HUWE1 in Treg / Th17 immune imbalance of immune thrombocytopenic purpura; 2) the specific mechanism of HUWE1 promoting Ets-1 ubiquitination degradation; 3) miR -125a-5p on the target gene HUWE1.We will further explore the molecular mechanism of HUWE1 on imbalance of Treg/Th17 imbalance, trying to provide a potential target for the treatment of ITP in the future.

免疫性血小板减少性紫癜（immune.thrombocytopenia，ITP）是以血小板减少和皮肤粘 膜出血为特征的自身免疫性疾病，Treg/Th17失衡发挥重要作用，但具体分子机制不清楚。前 期研究显示，ITP患者外周血CD4+T细胞中E3泛素连接酶HUWE1的表达增加；干扰HUWE1部分恢复 Treg/Th17平衡；HUWE1既是miR-125a-5p调控的靶分子，也可介导Ets-1的泛素化降解。由此 我们推测，miR-125a-5p表达下降使得HUWE1增加，降低Ets-1水平，在ITP发病中发挥重要作用。本项目重点研究：1）HUWE1在ITP.Treg/.Th17失衡中的作用；2）HUWE1促进Ets-1泛素化 降.解的具体机制；3）miR-125a-5p对HUWE1的调控情况。本项目的完成有助于阐明HUWE1在IT P患者Treg和Th17失衡中的内在机制，并提供一个直接的治疗靶点。

背景：免疫性血小板减少症（ITP）是一种获得性自身免疫、出血性疾病，大量研究表明Th17/Treg失衡参与了ITP的发展过程。越来越多的证据表明长链非编码RNA（lncRNAs）与多种自身免疫性疾病相关，miRNA亦被证实参与ITP的发病。本研究探讨了miR-199a-5p、miR-106b-5p、lncRNA GAS5、HUWE1在ITP中Th17或 Treg细胞分化的作用。.主要研究内容：利用CD41敲除建立ITP小鼠模型；采用qRT-PCR检测GAS5、miR-199a-5p、miR-106b-5p；流式检测Th17细胞、Treg细胞在CD4+T细胞中的百分比；ELISA测定IL-17和IL-10的水平；Western blotting检测RORγt、Foxp3、STAT3；RNA pull-down、RIP分析和泛素化测定等方法，证实miR-199a-5p、miR-106b-5p、lncRNA GAS5、HUWE1在Th17/Treg失衡中的作用，从而影响ITP的发病。.重要结果：GAS5通过促进TRAF6介导的泛素化加速STAT3的降解抑制Th17细胞分化；miR-106b-5p通过NR4A3 / Foxp3途径调节ITP中Treg / Th17的免疫失衡；miR-199a-5p通过控制脂肪间充质干细胞(ADSCs)获得的细胞外小泡(EV)抑制Th17分化从而缓解ITP；HUWE1促进Ets-1蛋白的泛素降解，从而抑制Treg细胞的分化，而抑制HUWE1可减轻小鼠ITP。.科学意义：ITP是一种自身免疫介导的获得性出血性疾病，可以发生在儿童和成人。在儿童中，大多数ITP是一种自限性疾病，但仍会有部分儿童会转变成慢性、难治性ITP，给患者及家庭带来身体伤害及心理、经济压力，本研究通过实验证实miR-199a-5p、miR-106b-5p、lncRNA GAS5、HUWE1在ITP发生的作用，为难治性、慢性ITP找到潜在治疗靶点。