PRMT1参与的炎症相关miRNA调控网络在气道炎症中的机制研究

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.

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。

蛋白质精氨酸转甲基酶（PRMTs）家族在哮喘的上皮细胞和成纤维细胞的功能调节中起着关键作用。本项目的支持，团队研究证实了PRMT1和miRNA调控网络参与气道炎症的机制，为哮喘的防治提供理论和实验依据。 研究结果如下：（1）在肺上皮细胞BEAS-2B中过表达PRMT1 48h进行small-RNA seq，将差异表达基因与肺炎相关的5个在线数据集差异表达sncRNAs比对后，发现了23种PRMT1调控的肺部炎症相关sncRNA。（2）肺上皮细胞中，PRMT1的异常表达可以显著影响多种炎症相关成熟miRNA表达；细胞因子TGF-β1可以激活PRMT1对miRNA的调控；PRMT1作为共转录因子与转录因子STAT1和RUNX1形成转录共激活物，进一步特异性的结合并激活pri-let-7i，从而调控成熟let-7i的表达。（3）miRNA23a在急性及慢性AIPI模型肺组织中表达升高，生物信息学预测的miRNA23a的靶基因BCL2及CXCL12在肺组织中表达均显著降低；细胞学实验证实上皮细胞中IL-4调控miRNA23a表达升高，而成纤维细胞中miRNA23a的高表达由TSLP调控。研究明确了miR-23a 通过在上皮细胞和成纤维细胞中的通过不同的靶基因调控气道炎症和气道重塑过程。（4）TGF-β能够通过SMAD2/3→C/EBPb→PRMT1信号通路介导成纤维细胞中PRMT1的表达升高，进一步调控使PGC-1a的表达升高，从而参与成纤维细胞中线粒体含量的增多，并参与了TGF-β诱导的成纤维细胞向肌成纤维细胞转化的过程。（5）明确了维生素D添加的不同策略对肺部炎症反应的作用及机制。单次维生素D3预处理通过降低STAT3蛋白磷酸化、上调ACE2蛋白表达、提高Ikb-α蛋白磷酸化和改变肺微生物群组成，显著减轻LPS诱导的肺部炎症。然而，长期补充维生素D3没有效果。此外，肺微生物群中啮齿类杆菌的丰度与IL-1β、IL-6和TNF-α的表达呈正相关。这些发现阐明了维生素D3可能通过调控肺部微生物组成从而预防或缓解肺部炎症疾病。综上，项目完成了PRMT1通过调控炎症相关pri-miR的表达参与气道炎症的研究，并在此基础上拓展了PRMT1、肺部菌群在炎症和气道重塑中的作用机制，为哮喘的防治及新药研发提供实验基础和理论依据。