IL-17通过miRNA调控小鼠重症哮喘发病作用机制的研究

IL-17 plays an important role in the pathogenesis of severe asthma, but the involved mechanism has not been elucidated. The literature and our preliminary findings suggest that miRNA regulation is an important mean for IL-17 in disease progression. On the basis of selection of miR-207 and miR-5112 related with IL-17 in severe asthma, the project intends to investigate the regulatory role of miRNA for IL-17 in severe asthma and the involved mechanisms. Firstly, we will further clarify the relationship between IL-17 and miRNA expression in disease, the localization of dysreulated miRNA expression will be investigated by in situ hybridization, laser capture microdissection technique and q-PCR. The regulation of target genes by miRNA will be detected by the transfection of miRNA target gene luciferase reporter plasmid towards cells. Secondly, by using the cells transfected with miRNA mimics or inhibitors, the effects of miRNA on the differentiation of Th2 cells, airway smooth muscle cell proliferation and secretion, NF-KB and PI3K signaling pathway will be investigated. Finally, the influence of in vivo miRNA intervention on the pathogenesis of severe asthma and its mechanisms will be investigated on the mouse model of severe asthma. The project is a new development of combination of severe asthma pathogenesis studies and miRNA research, which will make a useful attempt to explore the potential miRNA regulatory means.

IL-17是重症哮喘中的重要致病因子，其作用通路尚未阐明，已有文献和本项目组前期研究结果提示miRNA是IL-17调控疾病进展的重要手段。本项目拟基于前期筛选得到的在重症哮喘中与IL-17相关的miR-207和miR-5112，研究IL-17通过miRNA对重症哮喘的调控及机制。首先，将进一步明确疾病中IL-17对miRNA表达的影响，通过原位杂交与激光捕获显微切割技术和q-PCR定位miRNA的差异表达，并通过细胞转染miRNA靶基因荧光素酶报告质粒明确其对靶基因的调控；其次，采用细胞转染miRNA模拟物或抑制物，研究其对Th2细胞分化、气道平滑肌细胞分泌与增殖以及NF-KB和PI3K信号通路的调控作用；最后，通过小鼠重症哮喘模型探讨在体干预miRNA表达对重症哮喘发病的影响及机制。本项目是重症哮喘发病机理研究与miRNA研究结合的新发展，为探讨以miRNA为潜在调控手段做出有益的尝试。

哮喘是一种以可逆性气流受限为特征的气道慢性炎症性疾病，发病率和死亡率呈现上升趋势。哮喘中对患者健康生命威胁最大的是重症哮喘，到目前为止，临床仍缺乏有效治疗措施。新近研究以及本项目组前期研究结果表明，Th17细胞数量及其分泌的细胞因子IL-17A/F在重症哮喘发病中起重要作用。miRNA在哮喘发生发展中的作用已日益受到关注，研究显示一些miRNA在哮喘患者中表达失调。但由于研究时间短，与IL-17相关的miRNA对重症哮喘的调节作用及其机理尚未阐明。前期工作中我们对IL-17基因缺失小鼠重症哮喘模型进行了miRNA表达谱分析，发现miR-365-3p低表达与IL-17致病作用相关。本项目中我们对IL-17通过miR-365-3p在重症哮喘中起致病作用及机制进行了深入研究。首先，miR-365-3p在小鼠肺组织中主要表达于气道平滑肌细胞和炎症细胞中，重症哮喘模型小鼠上述细胞中的miR-365-3p表达明显减低。ARRB2和PIK3R3为其中与哮喘发病相关的靶基因。体外给予小鼠肺泡支气管上皮细胞IL-17刺激，发现IL-17可显著降低miR-365-3p的表达水平，同时ARRB2和PIK3R3的表达水平增高。其次，miR-365-3p抑制物能够促进气道平滑肌细胞炎症因子的表达和增殖，同时激活PI3K通路。最后，miR-365-3p的模拟物可使重症哮喘模型小鼠气道阻力下降，炎性细胞浸润减低，胶原蛋白沉积减少，气道平滑肌面积减少。本项目是对重症哮喘中IL-17通过miRNA对发病关键环节起作用的首次探索，是重症哮喘发病机理研究与miRNA研究结合的新发展，为探讨以miRNA为潜在调控手段做出了有益的尝试。