环状RNA介导狼疮易感位点参与疾病炎症通路调控的机制研究

Most disease-associated SNPs of autoimmune diseases identified by GWAS are located in the noncoding regions of genome and now exploring the role of these SNPs in the development of autoimmune diseases is a hot area. Our previous work has found the mechanism that miRNA-mediated susceptible SNP involved in the pathogenesis of lupus. In this project, we originally studied the role of circular RNA in the pathogenesis of inflammatory injury in lupus, and preliminary findings show that circTNPO3, located in lupus susceptible region, is highly expressed in lupus patients, thus involving in the regulation of inflammatory factors expression such as IL-6. So we propose a hypothesis that in lupus patients, functional SNPs affect the expression of circTNPO3 by influencing the binding of transcription factor GATA1; further, circTNPO3 regulates the function of miRNA or RNA binding protein and participates in the development of the disease. This project aims to elucidate the role of circTNPO3 mediating the development of the disease by interaction with susceptible SNPs. And we will apply strategies, such as gain-of-function and loss-of-function, knock-out, transcriptional regulation, molecular interactions and so on, to investigate the mechanisms. The expected results of this project will help to understand the pathogenesis of SLE and lay the foundation for individualized diagnosis and treatment of the disease.

遗传学研究发现的自身免疫病的绝大部分易感位点位于基因组非编码区域。目前探索这些位点在疾病发生发展中的作用是这个领域的研究热点。本课题组前期发现了miRNA介导疾病遗传易感位点参与狼疮关键致病通路激活的机制。在此基础上，我们原创性研究环状RNA参与狼疮炎症损伤的作用机制，初步发现狼疮患者中高表达的circTNPO3参与了IL-6等炎症因子的表达调控。本项目基于前期研究基础提出以下假说：在狼疮患者中，功能性的SNP通过转录因子GATA-1来促进circTNPO3的产生，circTNPO3可以通过影响miRNA（或RNA结合蛋白）的功能参与炎症通路的过度激活从而参与疾病的发生发展。本项目拟通过功能获得和功能缺失策略，结合转录调控、信号传导和分子相互作用研究来解析circTNPO3介导遗传易感位点参与疾病发生发展的作用机制。该项目的预期成果将有助于理解SLE的发病机制，为疾病的个体化诊疗打下基础。

本课题主要探究在SLE病人中异常表达的环形RNA circTNPO3在先天免疫通路中的作用及其对SLE发病的贡献，为SLE发病机制研究提供新层面的认识。我们首先对SLE病人和健康人外周血单个核细胞（Peripheral blood mononuclear cells, PBMC）来源的单核细胞、B细胞和T细胞亚群进行转录组学分析，发现在SLE不同亚群细胞中有大量异常表达的circRNAs。我们着眼于SLE疾病遗传易感区域IRF5-TNPO3，并通过qRT-PCR 进行验证，发现circTNPO3在病人单核细胞中异常高表达。通过对circTNPO3进行敲低或者过表达的干预，我们发现circTNPO3在单核细胞中能够正向调控LPS诱导的炎性细胞因子IL-6、IL-12等和细胞表面呈递分子HLA-DRA、CD80等的表达水平。我们进一步对circTNPO3敲低的细胞进行RNA-seq，通路富集分析表明circTNPO3主要调控TLR（Toll-like receptor, TLR）通路的活化。通过Western Blot我们验证了敲低circTNPO3能够下调LPS诱导的ERK、p38和p65等的磷酸化水平。机制上，circTNPO3定位于细胞质中，通过吸附miR-26a和miR-26b等miRNAs分子减少对TLR通路上游的MEKK2、TAB2和PAK1等分子的靶向，进而促进LPS诱导的TLR通路的激活。过表达circTNPO3可以逆转miR-26靶向TLR通路导致IL-6、IL-12和 CD80等的下调。在SLE病人单核细胞中，circTNPO3的表达水平与IL-6呈明显正相关，敲低circTNPO3可以下调SLE病人单核细胞中炎症因子的水平。该项目揭示了一个在SLE病人中异常高表达circTNPO3能够作为miRNAs的“海绵”吸附miR-26a和miR-26b，来调控单核细胞中TLR信号转导通路中的活性，进而调节了LPS诱导的炎症因子和细胞表面分子的表达。CircTNPO3在SLE病人中的过度表达可能是导致病人体内一些细胞因子异常高表达的因素。我们的研究为揭示SLE发病机制开辟了一个新的视角，这为SLE的临床诊断和治疗提供了新的方向。