miR-301a对炎症性肠病Th1和Th17细胞增殖分化的免疫调节作用

Idiopathic inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are chronic inflammatory disorders of the gastrointestinal tract. Abnormal immune responses in intestinal mucosa toward commensal bacterial flora together with persistent mucosal infection, intestinal mucosal barrier defects, genetic and environmental factors may be associated with the etiology and pathogenesis of human IBD. MicroRNAs (miR) are small (approximately 18-22 nucleotides), non-coding RNAs that posttranscriptionally regulate gene expression by binding to the 3'-untranslated region of target mRNAs, leading to mRNA degradation or translational inhibition. miR-301a has been found to be highly expressed in T cells, which targets the NF-kB and IL-6-STAT3 signaling pathways and regulates the proliferation and differentiation of Th17 cells. In our preliminary experiments, we have found that miR-301a is markedly increased in peripheral blood mononuclear cells and inflamed mucosa of IBD patients, and facilates Th1- and Th17-mediated immune response in intestinal mucosa. Thus, miR-301a is supposed to be involved in the development of inflammation in intestinal mucosa, particularly in human IBD. In this study, we will study the expression of miR-301a in peripheral blood and inflamed mucosa from IBD patients, particularly from CD patients treated with anti-TNF monoclonal antibody (e.g., infliximab). We will also investigate the potential role of miR-301a in regulating T cell immune responses in intestinal mucosa, and try to determine the potential target genes of miR-301a. Experimental colitis models in mice will be established in the laboratory and treated through blockage of miR-301a in vivo to determine whether it could prevent intestinal mucosal inflammation. This work will clarify the potential role of miR-301a in the pathogenesis of IBD, and provide an important theoretical basis for IBD immune therapy.

炎症性肠病（IBD）的病因和发病机制可能与肠黏膜免疫调节异常、持续肠道感染、肠黏膜屏障缺损、遗传和环境等因素有关。microRNA（miR）是一类非编码的内源性小分子RNA，通过结合靶基因的3'-UTR端，调控相关靶基因的表达和功能。研究报道miR-301a表达在T细胞上，对NF-kB、IL-6-STAT3信号，以及Th17细胞效应有密切调节作用。我们前期研究发现IBD患者外周血与肠黏膜组织内miR-301a表达升高，并促使肠黏膜组织Th1和Th17细胞效应应答。据此提出miR-301a可能参与肠黏膜炎症发生过程。本课题着重研究miR-301a在IBD中的表达和对肠黏膜组织CD4+ T细胞免疫调节效应，寻找miR-301a的靶基因，阐明其在IBD发生过程的免疫调节作用。建立小鼠慢性结肠炎模型，靶向阻断miR-301a效应应答，观察肠黏膜炎症发展变化，为临床上治疗IBD寻求新的治疗靶点。

炎症性肠病（IBD）包括克罗恩病（Crohn's disease, CD）和溃疡性结肠炎（ulcerative colitis, UC），是发生在胃肠道的慢性非特异性炎症性疾病。IBD的发生原因不明，可能是肠黏膜免疫系统对肠道微生物抗原异常免疫应答引起。microRNA（miR）是一种非编码RNA，靶向调节靶基因的功能。本课题重点研究在IBD肠黏膜炎症发生时，miR-301a对IBD患者肠黏膜组织内Th1和Th17细胞增殖分化的免疫调节作用。我们研究发现miR-301a在活动期CD和UC患者炎症肠黏膜组织内表达比缓解期患者肠黏膜组织和健康者肠黏膜组织表达明显升高，其主要表达在CD4+ T细胞和肠上皮细胞上。体外培养外周血CD4+ T细胞，使用anti-CD3和anti-CD28 mAb刺激，使用不同细胞因子（TNF-a、IL-17A、IL-6、IL-23、IFN-g、IL-10、IL-12）辅助刺激，发现TNF-a可显著上调miR-301a表达，IL-23和IL-6也有一定上调效应，而其他细胞因子对miR-301a的表达无作用。体外培养CD和UC患者外周血CD4+ T细胞，使用表达miR-301a的慢病毒转染，发现可刺激IBD CD4+ T细胞表达高水平IL-17A、RORC和TNF-a mRNA，而使用表达anti-miR-301a的慢病毒转染可显著降低CD4+ T细胞表达这些分子。我们使用双荧光素酶报告基因方法证实了SNIP1和BTG1为miR-310a的靶基因。使用TNBS灌肠诱导BALB/c小鼠发生实验性结肠炎，使用antisence miR-310a灌肠治疗，发现antisence miR-310a治疗的小鼠结肠炎症明显缓解。进一步构建了miR-301a基因敲除（miR-301a-/-）小鼠，使用2% DSS饮用水喂养小鼠诱导急性结肠炎发生，发现miR-301a-/-小鼠结肠黏膜炎症显著降低，同时发现结肠炎发生癌变的程度也明显降低。本研究阐明了miR-301a在肠黏膜炎症发生过程中起着重要的促炎症效应，通过靶向阻断miR-301a有可能用于临床上治疗IBD的发生发展。