特应性皮炎血浆外泌体传递hsa_circ_0001071靶向miR-146a-3p抑制Treg细胞功能的作用及分子机制

Treg cells play an important role in the pathogenesis of AD because of their negative immune regulation. The impaired function of Treg cells is an important reason for the occurrence and development of AD, but the mechanism has not been fully elucidated. It has been proved that miR-146a is highly expressed in Treg cells. Removal of miR-146a can destroy the immunosuppressive function of Treg cells. In our previous work, high-throughput microarray screening combined with bioinformatics analysis revealed that the highly expressed circRNA "circ_0001071" in plasma exosomes of AD patients may target and compete to bind to miR-146a-3p through the "molecular sponge" mechanism, preventing the binding of miR-146a-3p to the target gene STAT1mRNA, thus playing an important role in the regulation of Treg cell function. On the basis of the previous work, this project aims to clarify the role of AD plasma exosome in regulating Treg cell function through Treg suppression assay, using FISH, immunoprecipitation, pull-down, PCR, Western blotting and other experimental techniques, and also reveal the role of has_circ_0001071 overexpressed in AD plasma exosome in regulating Treg cell function and its molecular mechanism, so as to provide new targets for future possible therapeutic strategies.

Treg细胞功能受损在特应性皮炎（AD）发生发展中起到重要作用， 是研究热点之一。既往证明，miR-146a在Treg细胞中高表达，去除miR-146a可破坏Treg细胞免疫抑制功能。我们前期工作中通过高通量芯片筛选结合生物信息学分析发现，AD患者血浆外泌体中高表达的环状RNA“circ_0001071”可能通过“分子海绵”机制靶向竞争性结合miR-146a-3p，阻止miR-146a-3p与靶基因STAT1 mRNA结合，进而在Treg细胞功能调控中发挥重要作用。本课题拟在前期工作的基础上，通过细胞水平的研究，明确AD血浆外泌体调控Treg细胞功能的作用，采用FISH定位、免疫共沉淀、pull-down、PCR等实验技术，揭示AD血浆外泌体通过高表达has_circ_0001071调控Treg细胞功能的分子机制，明确其在介导AD免疫紊乱及发病中的意义，为未来可能的治疗策略提供新靶点。

本研究通过高通量芯片筛选结合生物信息学分析发现，AD患者血浆外泌体中高表达环状RNA“circ_0001071”，该分子可通过“分子海绵”机制靶向竞争性结合miR-146a-3p，后者在Treg细胞中高表达，去除miR-146a可破坏Treg细胞免疫抑制功能。将AD患者血浆外泌体与Treg细胞共孵育，通过PCR、Western、ELISA检测，发现STAT1、Smad7表达上调、Smad2和Smad3磷酸化抑制、细胞因子TGFβ和IL10表达下调。荧光素酶报告实验证实has_circ_0001071/miR-146a-3p、miR-146a-3p/STAT1可互相结合，一定程度上证实has_circ_0001071/miR-146a-3p/STAT1之间的互作关系，初步明确AD患者血浆外泌体可能通过向 Treg 细胞传递关键的 has_circ_0001071 分子，通过分子海绵作用靶向与miR- 146a-3p结合，竞争性抑制其与靶基因STAT1 mRNA的结合，从而减弱miR-146a对STAT1表达的抑制，上调STAT1的表达。磷酸化STAT1通过促进Smad7表达，抑制Smad2和Smad3的磷酸化，阻止炎症抑制因 子TGF-β,IL10信号转导，来实现对Treg细胞的调控，为AD的发病机制和开发治疗新靶点提供了理论基础。

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