miR-154上调Wnt/β-catenin信号通路在肺纤维化中的作用及机制研究

Myofibroblast formation is the important step in the lung fibrosis development. Studies show miR-154 increase migration and proliferation of lung fibroblasts, the increase in proliferation induced by TGF-β1 was not observed when normal human lung fibroblasts or IPF fibroblasts were transfected with a miR-154 inhibitor. inhibitors of the Wnt/β-catenin pathway, reduced the proliferative effect of miR-154.Our first stage study and documents proved activation and upregulation of Wnt/β-catenin signal pathway and miR-154 in lung fibrosis tissue.The online biological information study analysis indicated that miR-154 participate in the regulation of Wnt/β-catenin signal pathway. According the information,we conclude that the role of upregulation of the Wnt/β-catenin pathway by miR-154 is very important in lung fibrosis.In this study, We will treat the lung fibroblasts with TGF-β1 and miR-154 agomirs or mir-154 antagomirs, study the change of Wnt/ β-catenin signal pathway. Then use ICG-001 to inhibit Wnt/β-catenin signal pathway, analyze the effection; We will also use bleomycin to induce mice lung fibrosis, study the role of miR-154 in lung fibrosis. To elaborate the mechanism of miR-154 upregulating Wnt/β-catenin pathway in lung fibrosis on cellular level; then test and verify the role of miR-154 in mice pulmonary fibrosis. The study will futher reveal the molecular mechanism of lung fibrosis, and explore new target spot during the treatment of lung fibrosis.

肌成纤维细胞生成是肺纤维化形成的重要一环，研究证实：miR-154可增强肺成纤维细胞的增殖和迁移能力，抑制Wnt信号通路后miR-154作用受到逆转。我们的前期研究及文献证实：Wnt信号通路和miR-154在肺纤维化组织中表达上调。生物信息学分析提示：miR-154参与调控Wnt/β-catenin通路。据此提出假设：miR-154上调Wnt信号通路在肺纤维化中起重要作用。本研究拟TGF-β1、miR-154等处理肺成纤维细胞，研究Wnt信号通路变化情况，验证miR-154对靶基因Dkk2的调控作用；用ICG-001抑制Wnt通路，研究其对miR-154作用的影响；并研究miR-154对小鼠肺纤维化的作用。从细胞水平阐述miR-154上调Wnt信号通路调控肺纤维化的机制，从动物水平验证miR-154在肺纤维化中的作用。该研究结果将进一步揭示肺纤维化的发病机制，为肺纤维化的治疗提供新靶点。

特发性肺纤维化（IPF）是一种慢性的进行性疾病，其特征是成纤维细胞对肺损伤的反应产生不受控制的细胞外基质沉积。尽管发现microRNA（miRs）表达失调与肺纤维化的病理过程有关，但对确切功能机制的理解仍然有限。在这项研究中，我们观察到从博莱霉素诱导的肺纤维化小鼠分离出的肺组织中，miR-154水平升高。此外，增加的miR-154水平与博来霉素（BLM）诱导的肺纤维化的严重程度增加以及成纤维细胞的异常分化和增殖有关。采用逆转录定量聚合酶链反应（RT-qPCR）和Western blot分析，确定DKK2为miR-154的靶基因，发现miR-154的表达变化改变了DKK2和β-连环蛋白在肺纤维化小鼠中的表达。此外，在体内和体外也证实了Wnt信号在肺纤维化发展中的重要作用以及ICG-001对Wnt通路的抑制作用。最后，用ICG-001逆转了在肺纤维化小鼠中由miR-154过度表达引起的纤维化增强。我们的研究结果表明，通过直接靶向DKK2调节Wnt信号通路，miR-154起到了促进作用，因此是一种新的肺纤维化治疗靶点。