LncRNA-ATB吸附miR-200c和miR-34c-3p调控EMT促进矽尘诱导肺纤维化的机制研究

Pneumoconiosis is the most common occupational disease in China. However, the pathogenesis is not clear which limits the breakthrough in effective treatment of pneumoconiosis. Epithelial mesenchymal transition (EMT) is one of the important events in silica-induced lung fibrosis since lung epithelium is a main resource of myofibroblast through EMT process. However, the molecular mechanism of EMT is still unclear. Our previous study indicated that lncRNA-ATB involved in regulation of EMT process. MiR-200c and miR-34c-3p were identified as potential targets of lncRNA-ATB by miRNA array screening and bioinformatics analysis, and verified by dual luciferase reporter gene together with RNA pull-down assay. Since the functions of single miRNAs have been reported broadly during lung fibrosis, study of combined effects of multi-miRNAs would be helpful to explore underlying mechanism network. With latest research achievements and our previous results, we hypothesize that lncRNA-ATB promotes EMT during the silica-induced pulmonary fibrosis by competitively binding with miR-200c and miR-34c-3p. To clarify the hypothesis, we will perform function recovery experiments by lncRNA-ATB plasmid together with miR-200c and miR-34c-3p mimic, or lncRNA-ATB siRNA with miR-200c and miR-34c-3p inhibitors during EMT, which could be helpful to determine the combination roles of lncRNA-ATB regulated miR-200c and miR-34c-3p. Meanwhile, the pathways that are possibly regulated by miR-200c and miR-34c-3p will be clarified. Also, over-expressed lentiviral vectors of miR-200c and miR-34c-3p will be constructed in vivo, separately or together, to reveal their combined effects in silica-induced pulmonary fibrosis. In addition, we will further verify the expression levels of lncRNA-ATB, miR-200c, and miR-34c-3p in serum and lung tissues of the patients with silicosis. Our results will be benefit to clarify the pathogenesis and provide new clue to molecular target therapy for pneumoconiosis.

尘肺病发病率高且机制不清，EMT是矽尘诱导肺纤维化的重要事件。前期研究发现lncRNA-ATB参与调控矽肺EMT进程，miRNA芯片、生物信息学预测和验证发现lncRNA-ATB可直接吸附miR-200c、miR-34c-3p。基于最新研究进展和我们的前期研究提出“lncRNA-ATB通过竞争性结合miR-200c、miR-34c-3p促进EMT调控矽尘诱导肺纤维化”的假设。拟用多种分子生物学手段在细胞水平研究lncRNA-ATB与miR-200c、miR-34c-3p对EMT的联合作用及其可能的分子机制；在动物水平通过单独和联合高表达miR-200c、miR-34c-3p进一步验证其抑制EMT对矽尘诱导肺纤维化的联合作用；在矽肺患者血清和肺组织中初步验证lncRNA-ATB与miR-200c、miR-34c-3p的表达。研究结果将有助于揭示尘肺病机制，为预防、治疗及疾病标志物提供线索。

尘肺病是由于长期吸入生产性粉尘引起的以肺组织不可逆性纤维化为主的全身性疾病，是我国每年新诊断职业病中最多的一类疾病，其中矽肺占了尘肺病中的40%左右，是进展最快、最严重的一种尘肺病。课题组紧跟国际前沿，用于先进的分子生物学理论及技术研究矽肺发病分子机制及干预靶点对尘肺病的预防及精准靶向治疗均具有重要的意义。.前期的研究发现，EMT是矽尘诱导肺纤维化的重要事件。课题组先检测了尘肺患者血清和TGF-β1处理的肺上皮细胞EMT模型中lncRNA-ATB水平，发现均显著上调。然后在细胞水平通过低表达lncRNA-ATB证实了其对EMT的调控作用。进一步筛检lncRNA-ATB在肺上皮细胞EMT中可能吸附或调控的miRNAs，分析和验证后发现miR-200c、miR-34c-3p、miR-29b-2-5p这3种miRNAs的种子序列与lncRNA-ATB存在潜在的结合区域，后续实验证实 lncRNA-ATB通过海绵吸附这三种miRNA（miR-200c、miR-34c-3p、miR-29b-2-5p）并影响其靶基因来调控EMT参与矽尘所致肺纤维化。研究结果提示lncRNA-ATB是肺纤维化的重要靶点。.课题组一直围绕非编码RNA调控矽尘诱导肺纤维化机制研究，力求为干预其发生发展提供科学依据。在以往的研究基础上，课题组对lncRNA，circRNA以及miRNA在矽肺发生发展的各个阶段和多种效应细胞中的作用及机制进行深入探索，为尘肺病的干预和治疗研究提供了新的思路和线索，推进了肺纤维化机制研究的进程。.课题组一直关注了矽肺发生发展过程中的主要细胞事件，并发现小窝蛋白-1（CAV1）及其7-氨基酸活性功能肽段CSP7能够调节成纤维细胞活化，这些发现为二氧化硅诱导肺的机理研究提供了新的思路。课题组还对接触硬金属粉尘会导致以呼吸道症状为特征的硬金属肺病 （HMLD）进行了探索，成功构建了硬金属诱导的急性肺损伤的小鼠模型并进行机制探索，进一步拓展了职业性粉尘导致肺部损伤的研究。