成纤维细胞-肌成纤维细胞转化介导的气道重塑过程中PRMT1靶蛋白的筛选鉴定及其功能研究

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. This occurs mostly through the release of inflammatory mediators, which elicit the proliferation, and activation of airway smooth muscle cells and fibroblasts and finally induces lung remodeling in chronic asthma. Fibroblasts-myofibroblast transition (FMT) is an important mechanism of airway remodeling in asthma. Our previous work found that the expression of protein arginine methyltransferase 1 (PRMT1) shifted from epithelial cells in acute disease stage to fibroblasts in chronic disease stage in E3 rat antigen induced pulmonary inflammation (AIPI) model, furthermore, TGF-β can induce PRMT1 and α-SMA upregulation in fibroblast, and the inhibition of PRMT enzyme activity by AMI-1 block the augment of α-SMA in TGF-β stimulated fibroblast. All of which implied that PRMT1 may play an important role in fibroblasts-myofibroblast transition induced remodeling process of chronic asthma. First, PRMT1 expression profile in lung tissue and lung primary fibroblast from healthy sujects and asthmatic patients will be screened by using real-time PCR, western blot and Immunohistochemistry. Then, the relationship between the PRMT1 expression, FMT phenotype changes and asthmaic parameters will be analyzed to find the proof that PRMT1 participating in FMT process of airway remodeling in chronic asthma. Second, 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, PRMT1 substrate protein and mutant 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 FMT process. Eventually, lenti-viral vector is employed for PRMT1 or substrate protein lower-expression in acute and chronic 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 effects of PRMT1 in FMT and airway remodeling in chronic asthma, and provide novel insights into understanding asthmatic pathogenesis.

我们发现蛋白质精氨酸甲基转移酶1（PRMT1）酶活性的抑制可显著降低TGF-β诱导的成纤维细胞中肌动蛋白α的表达，提示PRMT1可能参与成纤维细胞-肌成纤维细胞转化(FMT)介导的哮喘气道重塑过程。为证明PRMT1通过其转录后调控功能参与FMT，拟首先比较哮喘病人和正常人原代肺成纤维细胞的差异，观察PRMT1参与成纤维细胞的FMT过程；继而特异性甲基化免疫印迹联合质谱分析筛选PRMT1的靶蛋白，鉴定靶蛋白结构中甲基化的精氨酸残基位点，明确FMT过程中PRMT1甲基化的底物蛋白质；干预PRMT1的表达或酶活性，检测靶蛋白表达和甲基化改变，结合原代成纤维细胞的功能变化，分析PRMT1是否通过甲基化靶蛋白参与了FMT过程；最后，在体干预PRMT1或其关键靶蛋白，证实PRMT1和其靶蛋白参与了FMT介导的气道重塑过程。研究将确定PRMT1参与气道重塑的分子机制，为哮喘的防制提供理论和实验依据。

蛋白质精氨酸甲基转移酶1（PRMT1）酶活性的抑制可显著降低TGF-β诱导的成纤维细胞中α-SMA的表达，提示着PRMT1可能参与成纤维细胞-肌成纤维细胞转化(FMT)介导的哮喘气道重塑过程。因此，项目组针对 “PRMT1通过其转录后调控功能参与FMT”这一科学问题，利用对照和哮喘人群原代肺平滑肌细胞和成纤维细胞，初步证实了PRMT1参与了哮喘发生发展过程的调控机制及PRMT1在成纤维细胞FMT过程中的作用。.取得的主要代表性成果如下：.1. 阐明了气道平滑肌细胞中PRMT1稳定过表达的内在机制。PRMT1显著高表达于哮喘上皮细胞和上皮下组织，原代哮喘人肺平滑肌细胞较对照组显著高表达PRMT1；哮喘气道平滑肌细胞中mi-R19a的缺乏，导致MAPK1表达显著升高，哮喘平滑肌细胞中ERK-STAT1通路持续性活化，是导致哮喘平滑肌细胞PRMT1持续升高的原因，PRMT1酶活性的抑制可显著改善PDGF-BB导致的气道平滑肌细胞增殖及细胞外基质的分泌。.2. 证明了同一miRNA在成纤维细胞和上皮细胞中的差异性作用。无论在哮喘的急性炎症阶段或慢性重塑阶段，miR-23a表达显著升高，而miR-23a的靶基因CXCL12和BCL2均显著降低。在上皮细胞中，IL-4通过诱导miR-23a高表达而下调BCL2，而在成纤维细胞中，TSLP通过miR-23a而下调CXCL12的表达。.3. 首次揭示了热成形术对肺成纤维细胞线粒体功能的影响机制。支气管热成形术（bronchial thermoplasty，BT）是一种内镜下、微创性重症哮喘治疗方式。我们通过与瑞士Basel大学合作，获取了BT治疗前后病人原代细胞样本，发现BT治疗后，上皮细胞的增殖出现克隆化特征，BT治疗后培养的上皮细胞可显著降低气道下成纤维细胞的增殖和线粒体水平，C/EBPβ-ERK信号通路被抑制，治疗前后肺泡灌洗液蛋白组及上皮细胞转录组测序对比显示治疗后HSP60的蛋白及RNA水平均下降，HSP60重组蛋白可显著诱导肺成纤维细胞中PRMT1表达、ERK通路激活和细胞中线粒体功能。.至此，我们通过细胞、人群和动物在体水平三方面的系统研究，明晰了PRMT1在哮喘中的作用机理，丰富了PRMT1的功能学研究，为哮喘的预防和治疗提供了新的思路药物靶点，具有重要的理论和实践意义。.