miR-199a-5p介导克罗恩病Th17/Treg细胞分化失衡的机制研究

Crohn’s disease (CD), which is characterized by Th17/Treg imbalance，is an increasingly prevalent disease. Our previous study found that the expression of miR-199a-5p was greatly increased during the differentiation process of mice naïve T cells into Th17 cells and was greatly decreased during Treg differentiation in vitro. MiR-199a-5p expression was markedly overexpressed in CD CD4+ T cells from both PBMC and colonic biopsies. Via the Pearson’s correlation analysis, there was a positive relationship between miR-199a-5p and RORγt mRNA, and an inverse correlation between miR-199a-5p and FoxP3 mRNA in lamina propria CD4+ T cells. Results from bioinformatics prediction and luciferase report gene assay indicated that Caveolin-1 (CAV-1), a novel regulator of Th17/Treg differentiation, was the target gene of miR-199a-5p. Thus we hypothesize that miR-199a-5p modulates the differentiation of Th17/Treg cells by targeting CAV-1 and thereby promotes the pathogenesis of CD. In this project, human samples and the experimental colitis model will be used to prove that miR-199a-5p is involved in CD progression and the differentiation of Th17/Treg cells. We will further investigate the molecular mechanism that miR-199a-5p regulates the Th17/Treg cells differentiation by targeting CAV-1 through miRNA lentiviral vectors construction in vitro. The above results will shed a new light on understanding the pathogenic role of miR-199a-5p in the imbalance of Th17/Treg differentiation in CD and suggest that target therapy directly against miR-199a-5p may be a novel approach for the treatment of patients with CD.

克罗恩病（CD）是常见的自身免疫性疾病，Th17/Treg细胞分化失衡为其突出表现。我们前期发现：miR-199a-5p在体外诱导Th17细胞时表达升高，诱导Treg细胞时则表达降低；活动期CD患者外周血及肠粘膜固有层CD4+ T细胞中miR-199a-5p均显著高表达，且与RORγt mRNA水平呈正相关，与FoxP3 mRNA水平呈负相关；窖蛋白1（CAV-1）是miR-199a-5p的靶基因。而CAV-1是新近发现的调控Th17/Treg分化的因子，我们由此推测：miR-199a-5p通过靶向抑制CAV-1影响Th17/Treg细胞分化，参与CD发病。本课题拟通过临床标本及动物模型研究，获得miR-199a-5p参与CD发病及其与Th17/Treg分化相关的依据；并采用慢病毒过表达及干扰技术在体外研究miR-199a-5p对Th17/Treg细胞分化的调控机制，为CD防治提供新靶点。

克罗恩病（CD）在我国的发病率逐年增加，其病因和发病机制目前尚未明确，越来越多的观点认为其发病机制可能与肠粘膜对肠道异常微环境的免疫调节紊乱，肠粘膜屏障破坏等因素有关。miRNA是真核生物体内的小分子非编码单链RNA，已有研究证实miRNA参与肠粘膜稳态及CD炎症调节过程。对人CD患者的肠粘膜组织进行高通量miRNA测序发现miR-199a-5p表达较正常对照组增高，通过在人CD患者及小鼠TNBS模型中的qPCR验证我们发现miR-199a-5p在CD及小鼠肠炎模型中表达均增高，同时活动 期CD患者外周血及肠粘膜固有层CD4+ T细胞中miR-199a-5p均显著高表达，且与RORγt mRNA水 平呈正相关，与FoxP3 mRNA水平呈负相关。生物信息学软件预测窖蛋白1(CAV-1)是miR-199a-5p的靶基因，人CD患者及鼠TNBS模型中CAV-1表达均减低，与miR-199a-5呈负相关关系。但我们通过反复实验并未发现CAV-1表达与RORγt mRNA、FoxP3 mRNA具有相关关系，因此我们猜想是否miR-199a-5p靶向调控CAV-1影响肠上皮屏障功能而非Th17/Treg细胞分化。通过构建稳转株、qPCR、Western blot、荧光素酶报告基因等实验我们证实了在肠上皮细胞中，CAV-1是miR-199a-5p的靶基因，且miR-199a-5p通过靶向调控CAV-1破坏肠上皮紧密连接。上述调控关系在小鼠TNBS模型中得以验证。本课题通过体内及体外研究证实了miR-199a-5p在CD发病过程的可能作用，为CD临床治疗可能的新靶标提供实验依据。