miR-548在视神经脊髓炎中调控Th17细胞分化的机制研究

Neuromyelits optica (NMO) is a severe idiopathic inflammatory demyelinating disease in central nervous system, which always causes permanent disability in young people. It is suggested that epigenetic factors may play an important role in NMO development, and microRNAs (miRNAs) are one of the newly discovered epigenetic regulators. Our research suggested that CD58, a kind of immune-related cytokine, whose polymorphism is associated with the risk of NMO in Han Chinese. In NMO patients, there was down-regulated expression of miR-548, a SNP at the base of whose stem-loop was in strong linkage disequilibrium with the NMO-associated haplotype. Based on the genetic and pathway bioinformatics analysis and dual luciferase reporter assays, we speculate that miR-548 promotes pathogenic T helper 17 (Th17) cell differentiation through retinoic acid receptor-related orphan nuclear receptor (RORA) and is associated with NMO. In the present project, we plan to fully explore the role and pathogenesis of miR-548 in NMO by cellular and animal experiments. In the naïve CD4+ T cell, we will analyze the differentiation of Th17 cell, the expression of IL-17 and some Th17 cell lineage-specific transcription factors, after up-regulated expression or down-regulated expression of miR-548 and/or RORA. In order to further reveal that the influence of miR-548 on Th17 cell differentiation is related to the development and progression of NMO, we will prepare up-regulated expression or down-regulated expression of miR-548 in a mice model of NMO. We intend to observe the clinical manifestations, and the demyelination and inflammatory cells infiltration in the spinal cord at different stages. The differentiation of Th17 cell, the level of IL-17 and some Th17 cell lineage-specific transcription factors will also be measured. Overall, the results of this project will provide novel insights into the mechanisms of miR-548 contributing to the pathogenesis of NMO, and identify genes and pathways as potential targets for further treatment.

视神经脊髓炎(NMO)是一种严重的导致年轻人残疾的中枢神经系统炎性脱髓鞘疾病。我们前期工作发现免疫反应相关细胞因子CD58基因多态性和NMO患病风险相关，与多态位点紧密连锁的miR-548在NMO患者表达减少；结合生物信息学分析和双荧光素酶报告基因检测，推测miR-548可能作用于靶基因RORA调控Th17细胞分化，在NMO发病中发挥重要作用。本研究拟通过干扰幼稚T细胞miR-548表达，检测Th17细胞分化程度、IL-17和RORA等相关蛋白表达；通过干扰幼稚T细胞RORA表达，观察是否引起和miR-548表达上调或下调类似的分子免疫学改变，研究miR-548调控Th17细胞分化的机制；在动物水平，检测miR-548过表达或表达抑制的NMO小鼠不同时期脊髓神经元髓鞘破坏和炎症反应程度、Th17细胞分化相关蛋白分子改变，探讨miR-548调控Th17细胞分化对NMO发病的意义。

视神经脊髓炎谱系疾病(NMOSD)是一种严重的导致年轻人残疾的中枢神经系统炎性脱髓鞘的自身免疫性疾病。我们以往的研究发现免疫反应相关细胞因子CD58基因多态性和NMOSD患病风险相关。本研究通过qRT-PCR发现与多态位点紧密连锁的miR-548在NMOSD患者表达减少；参考文献和生物学信息筛选出miR-548可能的靶基因和相关的关键通路，寻找和Th17细胞分化相关的基因，并对候选基因进行双荧光素酶报告基因检测，发现RORA是miR-548的靶基因之一，推测miR-548可能作用于靶基因RORA调控T辅助细胞17(Th17)分化，在NMOSD发病中发挥重要作用。同期研究发现和健康人对比，病人体内naïve T细胞水平降低，Th17等发挥炎症作用的细胞在NMOSD患者体内比例增加，且T细胞分泌IFN-γ及TNF-α炎性因子也增加。通过对比病人及健康人外周血DNA甲基化模式差异，发现Th17通路异常富集。从NMOSD患者PBMC甲基化模式筛选出与Th17细胞分化相关基因Tespa1与Runx1，通过体外实验发现NMOSD组呈Runx1低甲基化、Tespa1高甲基化。差异甲基化位置在Runx1的1号外显子及Tespa1的启动子区。运用qPCR和分别用PMA+IONO(PI)兴奋性刺激或用CsA抑制性刺激细胞分离人外周血淋巴细胞，证实甲基化导致了两个基因表达降低，推测其进而调控Th17分化参与NMOSD的发病过程。双荧光素酶报告实验证实Runx1正调控下游RORγt及IL-17表达，直接促进Th17分化并参与自身免疫反应。我们的研究提示对miR-548调控NMOSD中Th17细胞分化并影响其功能这一问题进行研究，明确miR-548在NMOSD发病中的作用机制，为研发调控Th17细胞分化、治疗NMOSD的新药可提供一定的理论依据。而纠正Runx1异常甲基化或者合成选择性抑制Runx1功能的药物可能对NMOSD有治疗作用。