miR-21调控自噬活性在矽尘所致肺纤维化中的作用及其机制研究

Silicosis is the most serious occupational disease in China, with no effective treatment. The precise pathogenesis of silicosis may contribute to the development of novel threraputic strategies. Our preliminary studies suggested that silica caused a significant increase in miR-21 levels of murine lung tissues. LC3 II, an autophagic marker, was decreased in silica groups, whereas p62, an autophagic degration marker, was increased. Additionally, less autophagesomes were detected in the silica group than that in the control group. In the project, we hypothesized that miR-21 might regulate autophagy in silica-induced pulmonary fibrosis via PTEN-AKT-mTOR signal pathway. The intervention models of anti-miR-21, mouse PTEN sh lentiviral vector and rapamycin will be established to explore the mechanisms of silica-induce pulmonary fibrosis in vivo and in vitro. Fibrotic results and autophagic results will be collected to verify the hypothese that miR-21 might regulate autophagy in silica-induced pulmonary fibrosis via PTEN-AKT-mTOR signal pathway. Thus, understanding the exact mechanism underlying silicosis in the lungs may be aid to introduce treatment options to delay or reverse the progression of the disease. Moreover, miRNA and autophagy might exhibit therapeutic targets in silica-induced pulmonary fibrosis.

矽肺病是我国目前最严重的职业病，且尚无有效的治疗方法。深入探索矽肺病的发病机制有助于研发新的矽肺病治疗方法。前期研究结果表明矽尘可致小鼠肺组织miR-21表达增加，自噬活性标志物LC3 II表达增加，自噬体降解标志物p62表达降低，肺细胞自噬体减少。结合前期结果与文献报道，我们提出科学假设：miR-21可能通过调控PTEN-AKT-mTOR信号通路调节自噬活性影响矽尘所致的肺纤维化。本项目通过构建矽尘所致小鼠肺纤维化动物模型，初步明确矽尘所致肺纤维化中miR-21及自噬活性的改变情况；在整体动物水平及细胞水平，通过构建miR-21抑制剂\敲除PTEN慢病毒载体\自噬激活剂干预模型，分析肺纤维化及自噬活性相关指标，验证miR-21调控自噬活性在矽尘诱导肺纤维化中的作用及其机制。本项目不仅为全面理解矽肺病发病机制提供新的视角，也为以miRNA和自噬为靶点的矽肺病药物防治策略的应用提供理论依据。

矽肺病是由于长期吸入大量游离二氧化硅粉尘所引起，以肺部广泛的结节性纤维化为主的全身性疾病。矽肺病是我国影响面最广、危害最严重的职业病之一，目前尚无有效的治疗方法。矽肺病的发病机制尚未完全明确，因此深入探索矽肺病的发病机制有利于提出新的矽肺病治疗策略。研究报道miR-21可作为特发性肺纤维化的潜在治疗靶点，而其在矽尘所致肺纤维化中的作用少见报道。我们前期芯片结果显示矽尘所致小鼠肺纤维化动物模型中miR-21表达增加，且在后续实验证实，提示miR-21参与矽尘所致肺纤维化发生发展过程，但其作用机制尚不明。本项目采用体内实验构建矽尘诱导小鼠肺纤维化动物模型，通过病理切片、纤维化蛋白表达、自噬活性改变情况检测，揭示miR-21对矽尘所致小鼠肺纤维化过程的影响。体外实验通过双荧光素酶报告基因、蛋白免疫印迹等分子生物学实验，分析高/低表达miR-21后纤维化蛋白、自噬标志物及靶基因信号通路的改变，阐明miR-21的关键调控靶点及作用通路，揭示其影响矽尘所致肺纤维化过程的分子机制。另外，通过qRT-PCR方法检测矽肺病患者血清及肺组织中miR-21表达水平，均较对照组表达增加。结果表明miR-21在矽尘所致小鼠肺纤维化动物模型肺组织中表达增加，miR-21抑制剂可减轻矽尘所致小鼠肺纤维化表型，进一步机制探索发现miR-21可靶向作用于PTEN调控AKT/mTOR信号通路从而影响自噬活性参与矽尘所致肺纤维化的发生发展。本研究通过动物模型及细胞模型分析了miR-21在矽尘所致肺纤维化中的作用，阐明了miR-21通过PTEN/AKT/mTOR信号通路调控自噬活性影响矽尘所致肺纤维化的分子机制。研究结果有利于揭示矽尘引起肺纤维化的分子机制，有助于为矽肺病的防治提供理论依据，为提高职业人群生命质量做出贡献。