PRMT1参与的炎症相关miRNA调控网络在气道炎症中的机制研究

我们预实验发现肺上皮细胞中蛋白质精氨酸甲基转移酶1（PRMT1）的高表达能够改变多个炎症相关miRNAs及其pri-miR的表达，提示PRMT1可能通过影响炎症相关miRNA调控网络参与哮喘的气道炎症过程。本项目以PRMT1参与的炎症相关miRNA表达调控机制为核心，在比较抗原诱导的肺部炎症大鼠模型、哮喘病人和正常人原代肺上皮细胞PRMT1和miRNA谱差异的基础上，通过特异性甲基化免疫印迹联合质谱分析筛选PRMT1的底物蛋白，IP、慢病毒转染等鉴定PRMT1与底物蛋白的靶向关系，生物信息学分析、酵母双杂交、IP分析与PRMT1结合作用的转录因子，荧光素酶报告系统、EMSA、ChIP等鉴定底物蛋白或转录因子复合物对pri-miR的表达调控。最后，在体干预PRMT1或关键pri-miR，解析PRMT1和miRNA调控网络参与哮喘气道炎症的机制，为哮喘的防治提供理论和实验依据。

Asthma is a clinical syndrome characterized by recurrent episodes of airflow obstruction of variable duration and intensity, which may be triggered by viral infections and exposure to allergens or other environmental stimuli in predisposed individuals. The inflammation developed in asthmatic lung epithelial cells contributes to the pathophysiology of the disease. .Our previous work found that the expression of protein arginine methyltransferase 1 (PRMT1) participated the epithelial inflammation through IL-4/STAT6 signal pathway and related to esinophil infiltration, furthermore, in our pilot experiments, the upregulation of PRMT1 by recombinant plasmid induced elevated inflammatory miRNAs expression. All of which implied that PRMT1 may play an important role in inflammatory miRNAs regulation network and inflammation process of acute asthma. However, the transcriptional regulation of inflammatory miRNA itself, regulatory networks of miRNA and its molecular mechanism in regulating airway inflammation remain obscure. .To determine the molecular mechanism through which PRMT1 induced inflammatory miRNA expression, first, PRMT1 expression and miRNA profile in lung tissue and lung primary epithelial cell from healthy sujects and asthmatic patients will be screened by using miRNAseq, real-time PCR, western blot and Immunohistochemistry. The relationship between the PRMT1 expression, key miRNAs and inflammation phenotype changes and asthmaic parameters will be analyzed to find the proof that PRMT1 and key miRNAs participating in airway inflammation process in asthma.The substrate proteins of PRMT1 is detected by 2D ADMA specific immunoblotting and HPLC. The combine of PRMT1 and its substrates protein will be detected by pull-down assay, co-IP and yeast two hybrid. The vectors of PRMT1 and PRMT1 substrate protein will be constructed and expressed to find the catalyze sites of PRMT1 substrate protein. Upregulating or downregulating PRMT1 substrate protein expression using gene cloning or siRNA in vitro to confirm its effect on pri-miR expression. Second, bioinformatics analysis, co-IP and yeast two hybrid will be used to find PRMT1 combinded transcription factors.Then, the transcription elements will be identified in the key pri-miR gene promoter by way of bioinformatics analysis, luciferase reporter system, electrophoretic mobility shift, and chromatin immunoprecipitation assays. Eventually, lenti-viral vector is employed for PRMT1 or key pri-miR lower-expression in acute AIPI model in vivo to evaluate the therapeutic effectiveness and the corresponding molecular mechanism of the rescue for remodeling process of asthma. .In this project, we will reveal the PRMT1 regulated inflammatory miRNAs network and inflammation process of acute asthma, and provide novel insights into understanding asthmatic pathogenesis.