特发性皮肌炎外周血及肺脏microRNA表达谱及功能研究

Idiopathic dermatomyositis (DM) is an inflammatory autoimmune disease which mainly involving skin and skeletal muscle. East asian DM patients are at high risk of developping interstitial lung diseae (ILD) with unknown etiology, which often rapidly progresses to respiratory failure and the mortality is extremely high. Novel diagnostic tools and therapeutic targets are critically required. MicroRNAs (miRNAs) dysregulation can result in many autuimmune diseases, and its expression profiling and functional study have become useful tools in demonstrating intracellularly molecular regulation. This study aims to perform a high-flux screening of aberrant miRNAs profile in peripheral blood mononuclear cells and lung tissue on a pre-established miRNA study platform, which had successfully validated in the similar study of systemic lupus erythematosus and systemic sclerosis. The study will screen further on the miRNAs which regulating ILD-related inflammation and the expression of profibrotic cytokines through a large-sample clinical registry and biobank. The selected miRNAs will be validated in lung tissue of DM-ILD patients. The function of selected miRNAs on target genes will be studied and validated in mice lung epithelia. In addition, the lung epithilia will be processed by DM-ILD specific anti-MDA5 antibody and the change of selected miRNAs' expression will be detected, which will demonstrate the pathogenesis of DM-ILD by combining autoimmunity, epithelial injury and fibrosis.

特发性皮肌炎（DM）是一种累及皮肤、肌肉的炎症性自身免疫病，东亚患者易合并肺间质病变（ILD），其机制不清，常迅速进展为呼吸衰竭，死亡率极高，亟需提高诊断水平并探索新的治疗靶点。小分子RNA（miRNAs）调控异常可引起多种自身免疫病，其表达谱和功能研究已成为揭示细胞内分子调控机制的有力工具。本课题在已建立系统性红斑狼疮和系统性硬化症miRNAs表达谱的研究平台基础上，高通量筛选DM外周血单个核细胞和血浆中异常的miRNAs表达谱；通过大样本临床与标本库进一步筛选与肺部病变相关的调控炎症和促纤维化因子表达的miRNAs，并在DM-ILD患者肺组织中进行验证；利用小鼠肺上皮细胞和进行miRNAs对靶基因的功能研究和验证；采用对DM-ILD较特异的抗MDA5抗体处理肺上皮细胞，探索其在miRNA水平的促炎和致纤维化机制，试图在miRNAs水平将自身免疫、上皮细胞损伤和纤维化三方面机制联系起来，

特发性皮肌炎（DM）是一种累及皮肤、肌肉的炎症性自身免疫病，东亚患者易合并肺间质病变（ILD），其机制不清，常迅速进展为呼吸衰竭，死亡率极高，亟需提高诊断水平并探索新的治疗靶点。小分子RNA（miRNAs）调控异常可引起多种自身免疫病，其表达谱和功能研究已成为揭示细胞内分子调控机制的有力工具。本课题通过miRNA芯片技术高通量筛选DM外周血单个核细胞和血浆中异常的miRNAs表达谱，获得34条差异表达miRNA；通过大样本临床与标本库进一步筛选与肺部病变相关的调控炎症和促纤维化因子表达的4条miRNAs，并在DM-ILD患者外周PBMC和血浆中进行验证，首次发现了miR34a在DM组及合并ILD组表达显著增高，从而提示其可能参与DM-ILD的发病，并作为有潜在临床应用价值的DM-ILD生物标志物。..生物信息学分析提示miR-34a可能通过抑制其靶基因sirt1的表达，影响mTOR通路对纤维化过程的调控。mTOR信号通路是细胞内调节细胞增殖、生存、凋亡等重要功能的信号途径之一, 有研究提示mTOR信号通路异常可能与特发性肺纤维化发病相关，但具体机制尚不明确。在本研究中，我们构建了TGF-β1诱导的肺纤维化BEAS-2B细胞模型，并通过qRT-PCR和western blot检测肺纤维化标志物的表达水平对模型进行鉴定，发现α-SMA, N-cadherin, vimentin, collagen I的表达水平在TGF-β1加入后显著升高。进一步用mTOR抑制剂Rapamycin或激活剂处理TGF-β1诱导的BEAS-2B细胞，发现Rapamycin可以有效抑制TGF-β1诱导引起的纤维化相关蛋白的表达。利用miR-34a模拟物或抑制剂处理TGF-β1诱导的BEAS-2B细胞，发现miR-34a模拟物的加入可以抑制肺纤维化进程，同时发现miR-34a能抑制sirt1表达并降低mTOR的活性。进一步研究发现mTOR激活剂3BDO能逆转miR-34a模拟物对肺纤维化蛋白表达的抑制作用，表明miR-34a 可能通过介导mTOR调控肺纤维化。通过注射博来霉素诱导小鼠肺纤维化构建体内模型，结果表明博来霉素药物处理组小鼠肺组织出现严重的纤维化，纤维化因子的表达水平明显高于生理盐水对照组。将miR-34a和mTOR作为干预的靶点对炎症反应介导的肺纤维化具有治疗作用，而其中的具体分子机