miR-186参与Th17/Treg失衡及影响毛囊干细胞活化在重症斑秃中的机制研究

Alopecia areata is a T-cell mediated autoimmune disease. The latest research suggests the pivotal role of T-cell and hair follicle stem cells (HFSCs), however, little is known about the interaction between them. We previously revealed that the high expression of miR-186 both in peripheral blood CD4+ T cells and serum exosomes in patients with severe alopecia areata (AA) had a positive correlation with the percentages of Th17 cells. The expression of miR-186 targeted gene FoxO1 decreased in hair follicle bulge region (HFSCs) after handled with serum exosomes from AA patients. These results suggested that miR-186 might correlates with the imbalance of Th17/Treg cells and attenuation of hair follicle stem cell activity through exosomes. The present work aims to clarify the role of miR-186 in the T-cell differentiation and proliferation in severe AA and exosomal miR-186 in HFSCs attenuation. Furthermore, we seek to explore the novel therapeutic methods by experimental intervention on miR-186 and its related signal pathway in the C3H/HeJ mouse model of AA. The present work would contribute to clarify the pathogenesis and provide new thoughts for treatment of severe alopecia areata.

斑秃是T细胞介导的器官特异性自身免疫性疾病。最新研究提示T细胞和毛囊干细胞是重症斑秃发病、进展和治疗的关键，但至今关于T细胞和毛囊干细胞交互机制的研究仍较少。我们前期实验发现重症斑秃患者外周血CD4+T细胞和外泌体中miR-186显著上调，且与Th17数量正相关。重症斑秃患者血清外泌体处理毛囊隆突区(富含毛囊干细胞)可降低miR-186靶基因FoxO1的表达。这提示了miR-186参与调节Th17/Treg平衡，并通过外泌体途径作用于毛囊干细胞。本研究拟体外分离重症斑秃患者的T细胞并诱导其活化，通过转基因等分子实验手段阐明miR-186在斑秃T细胞分化扩增中的机制；并分离T细胞来源的外泌体诱导毛囊干细胞等实验验证外泌体miR-186影响干细胞活性；随后在斑秃小鼠上尝试通过干预miR-186及相关信号通路，探索重症斑秃新疗法。本项目的开展有利于阐明重症斑秃的发病机制并为治疗提供新思路。

斑秃是一种T细胞介导的器官特异性自身免疫性疾病。已有大量的研究证实miRNA广泛参与免疫相关疾病和炎症性皮肤病的发病过程，然而，关于miRNA在斑秃发病中调控作用的研究目前仍未见报道。本课题拟在前期研究基础上阐明下列几个问题： 1. 重症斑秃患者外周血miRNA的表达谱分析 2. 重症斑秃差异表达miRNA参与调控的靶基因及信号通路 3. miRNA是否参与调控斑秃外周血Th17/Treg失衡? 4. 差异表达miRNA及其靶基因是否是糖皮质激素治疗重症斑秃的作用靶点，能否作为病情活动性及预后的生物标记?.我们在活动期重症斑秃患者外周血中共鉴定出36个差异表达miRNA。靶基因预测及功能分析显示主要富集在一些信号通路。miR-186-5p是miRNA调控网络中的主要节点。差异表达的miRNA通过协同网络发挥调控作用，其中miR-185-5p、miR-125b-5p和 miR-186-5p可能在重症斑秃活动期发挥了重要的协同调控作用。我们用qRT-PCR和Western Blot检测斑秃患者CD4+T细胞miR-186-5p和FOXO1的mRNA及蛋白表达水平。采用荧光素酶报告系统证实miR-186-5p直接靶向FOXO1。斑秃患者CD4+T细胞中miR-186-5p表达显著上调, 而FOXO1的mRNA及蛋白表达水平均显著下调。正常人CD4+T细胞过表达miR-186-5p模拟物后FOXO1表达水平显著下降；斑秃患者CD4+T细胞过表达miR-186-5p抑制剂后FOXO1表达水平上升。斑秃患者CD4+T细胞过表达miR-186-5p抑制剂后，ROR-rt表达显著下降，FOXP3表达水平无显著变化；Th17相关细胞因子水平显著下降，Treg相关细胞因子无显著变化。重症患者外周血FOXO1 mRNA水平在治疗前和复发时显著下调,在糖皮质激素治疗后表达水平上升。外周血miR-210-3p、miR-1246、miR-186-5p水平在治疗前均显著高于对照组，但在治疗后及复发时表达水平无显著变化。.综上，我们的研究显示miR-186-5p靶向FOXO1促进斑秃患者外周血CD4+ T细胞向Th17细胞分化，参与调控斑秃外周血Th17/Treg失衡。患者外周血FOXO1 mRNA水平与病情活动性负相关，提示FOXO1可能是糖皮质激素的药物作用靶点之一。