COPD血清外泌体小RNA分子标志物的筛选及其在上皮-间质转化中的作用及机制研究

Chronic obstructive pulmonary disease (COPD) is a common disease of the respiratory system. Recent studies showed that there was the phenomenon of epithelial mesenchymal transition (EMT) in the airway epithelium of patients with COPD, and EMT is the main sources of airway muscle fibers. However, its early molecular markers and the pathogenesis of EMT are still not very clear. We found that changes of serum small RNAs were consistent with the small RNA variation in the lung tissue by RNA sequencing. Moreover, the exosome including the small RNAs in serum could significantly stimulate the formation of EMT in the A549 cells. Therefore, we hypothesized that the exosomes small RNA of serum may become a biomarker for early diagnosis of COPD and induce the EMT formation of epithelial cells and participate in the occurrence of chronic obstructive pulmonary disease. The study is designed to investigate whether the exosomes small RNAs of serum are associated with the severity of COPD by the patient and animal models through bioinformatics, RNA sequencing and histopathology method. At the same time, we observed the role and mechanism of serum exosome small RNAs in the epithelial mesenchymal transition. Finally, it is clear that the small RNA in the exocrine can be used as a biomarker for early diagnosis of COPD and its role and mechanism in epithelial mesenchymal transition and provides new targets and strategies for early diagnosis, treatment and prevention of COPD.

慢性阻塞性肺疾病(COPD)是呼吸系统的常见病和多发病，近年来研究显示COPD患者气道上皮存在上皮-间质转化(EMT)现象，且EMT是导致气道肌成纤维细胞的重要来源，然而有关COPD早期的分子标志物及EMT发生机制目前仍不十分清楚。我们前期通过RNA测序比对发现，早期血清中小RNA变化与肺组织呈现一致性，且血清携带小RNA的外泌体能够明显诱导A549细胞EMT发生，故提出血清外泌体小RNA可能成为COPD早期诊断标志物且诱导上皮细胞EMT发生参与COPD发展。本课题拟动物模型和COPD患者为切入点，通过生物信息学法、小RNA测序及组织病理学研究血清外泌体小RNA与病情严重程度的相关性；通过小RNA转染及RNAi 等技术观察其对上皮细胞EMT作用及机制。将有助于明确外泌体小RNA是否可作为COPD早期诊断标志物及其在EMT作用和具体分子机制，为COPD早期诊断、治疗及预防提供新的靶点和策略。

慢性阻塞性肺疾病(Chronic obstructive pulmonary disease, COPD) 是常见的呼吸系统疾病。近年来研究显示COPD患者气道上皮存在上皮-间质转化(epithelial mesenchymal transition，EMT)现象，然而有关COPD早期的分子标志物及EMT发生机制目前仍不十分清楚。我们前期通过RNA测序比对发现，早期血清中小RNA变化与肺组织呈现一致性，故提出血清外泌体小RNA可能成为COPD早期诊断标志物参与COPD发生、发展。研究表明，外泌体来源的miRNA能够调控重要的细胞活动，参与COPD的发生、发展，并能作为COPD的诊断标志物及潜在的治疗新靶点。本研究通过COPD动物造模，外泌体提取，生物信息学分析，细胞模型构建及COPD肺组织标本验证，分析外泌体小RNA是否可作为COPD早期诊断标志物及其在上皮-间质转化中的作用和机制，为COPD早期诊断、治疗及预防提供新的靶点和策略。本研究结果筛选COPD大鼠模型中差异表达的miRNA，共得到15个有差异表达的miRNA，其中表达上调的miRNA有8个，表达下调的miRNA有7个。通过临床肺组织标本收集及验证，提示外泌体源性的 miR-182-5p、miR-185-5p表达减少，同样使用香烟烟雾干预人支气管上皮细胞，筛选出的外泌体源性的miR-182-5p、miR-185-5p表达也明显减少，提示它们可能参与了COPD的发生、发展。进一步的细胞试验提示差异表达的miRNA可能通过支气管上皮细胞的上皮-间质转化参与COPD的发生、发展。总之，本研究筛选并验证了COPD大鼠血清外泌体中差异表达的miRNAs，并对差异表达miRNA靶基因采取GO富集及KEGG富集分析，探索了COPD的发生、发展的信号通路，为阐明COPD的发病机制及提供潜在的治疗新靶点提供理论基础。