LncRNA(PFL)通过miR-146b/26a调控EMT参与特发性肺纤维化的机制研究

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and high lethality fibrotic lung disease with poor treatment and unknown etiology. More and more evidences indicate that long non-coding RNAs (lncRNAs) play important roles in the process of cancer and cardiovascular diseases. However, the role and mechanisms of lncRNAs in the progression of IPF is poorly understood. In the previous studies, we found that Yap1, a key downstream gene of Hippo pathway, could induce epithelial-mesenchymal transition (EMT) in cultured AECⅡ cells. In addition, miR-146b/26a (miR-146b and miR-26a), which are regulators for Yap1/HMGA2, are decreased in pulmonary fibrosis. Further study indicates that lncRNA-PFL was up-regulated in the lungs of mice with experimental lung fibrosis, and lncRNA-PFL has the potential binding sites for miR-146b/26a. We thus assume that PFL induces EMT by regulating the miR-146b/26a-Yap1/HMGA2 pathway, and then causes the occurrence of IPF. To address this hypothesis, we will perform the following experiments: 1) to reveal the change of lncRNAs and Hippo pathway in IPF; 2) to evaluate the role of PFL on EMT and IPF; 3) to examine the effect and mechanism of PFL on miR-146b/26a; 4) to elucidate the underlying mechanism of miR-146b and Yap1 in the process of EMT and IPF; 5) to investigate the potential therapeutic role of targeting PFL or Yap1 in IPF. This project can not only expand the understanding of the mechanism and therapeutic strategy of IPF, but also provide a theoretical basis for the research and development of new drug for pulmonary fibrosis.

特发性肺纤维化（IPF）发病机制不明确，尚无理想防治措施。长链非编码RNA (lncRNA)是肿瘤、心血管等疾病的关键调控分子，但其在IPF中的作用仍不清楚。前期发现：Hippo通路关键基因Yap1诱导EMT发生；miR-146b/26a在IPF中表达降低且预测调控Yap1/HMGA2；lncRNA-PFL在IPF中表达升高并具有miR-146b/26a结合位点。由此，我们假设：PFL通过miR-146b/26a-Yap1/HMGA2诱发EMT，导致IPF的发生。拟研究：1）lncRNA及Hippo通路在IPF中的变化规律；2）PFL对EMT及IPF的调控作用；3）PFL对miR-146b/26a的调节作用；4）miR-146b、Yap1参与EMT及IPF的分子机制;5）靶向PFL、Yap1对IPF的治疗作用。本项目将完善并拓展我们对IPF发病机制的认识，为新药研发提供理论依据和治疗靶点。

特发性肺纤维化（IPF）被称为“不是癌症的癌症”，其致死率高，预后差，但其发病机制仍不明确，缺乏理想的治疗手段。长链非编码RNA (lncRNAs)是肿瘤、心血管等疾病的重要调控分子，但其在IPF中的作用尚不清楚。本项目（1）基于芯片筛选和生物信息学分析，筛选出在肺纤维化中差异表达的多个lncRNAs，选取变化最为显著的3个lncRNAs（NONMMUT065582，PFAR；NONMMUT021928, PFAL；NONMMUT022554, PFRL）开展研究，发现分别过表达lncRNA PFAR/PAFL/PFRL均可激活肺成纤维细胞，增加胶原沉积，而沉默这三个lncRNA则发挥相反作用；使用腺相关病毒敲减lncRNA PFAR/PAFL/PFRL可减轻BLM诱导的肺纤维化。进一步研究发现，lncRNA PFAR/PAFL/PFRL通过ceRNA机制分别抑制miRNA-138、miRNA-18a、miRNA-26a表达及活性，使其下游靶基因YAP1、CTGF、smad2表达升高，进而发挥对肺纤维化的调控作用。（2）发现过表达Hippo通路核心基因YAP1可促进肺成纤维细胞活化，而敲减YAP1抑制TGF-β1引起的成纤维细胞活化并显著减轻BLM诱导的小鼠肺纤维化；进一步研究表明，YAP1通过与TEAD结合转录调控Twist表达进而发挥促纤维化作用；在此基础上，采用生物信息学分析GEO数据，筛选在IPF中差异表达的miRNAs，构建miRNA-Hippo通路调控网络。（3）探索Hippo通路抑制剂的抗纤维化作用：发现Verterporfin可抑制肺成纤维细胞活化；褪黑素通过与其受体结合影响Hippo通路，调控YAP1表达及其核转位，最终抑制成纤维细胞活化及小鼠肺纤维化；同时发现褪黑素可通过调控Apelin改善BLM诱导的肺泡上皮细胞线粒体功能紊乱，保护肺泡上皮损伤，减轻BLM诱发的肺损伤。（4）揭示lncRNA对EMT的调控作用，发现YAP1通过结合TEAD转录调控Slug表达促进EMT，干扰lncRNA PTAR/PTAF或YAP1可抑制EMT进程，并在此基础上构建了Hippo通路及EMT调控网络数据库，为伴EMT相关疾病的研究提供参考。以上结果不仅拓展了我们对肺纤维化发病机制的认识，为肺纤维化的防治提供了新的理论依据，并且有助于促进新型抗纤维化药物的研发。