Extracellular vesicles (EVs) play a vital role in normal lung physiology to maintain homeostasis in the airways via intercellular communication. EVs include exosomes and microvesicles, and are characterized by their phospholipid bilayers. EVs have been recognized as novel circulating biomarkers of disease, which are released by different cell types. In this study, we used different EV isolation and purification methods to characterize the plasma-derived EV miRNAs from non-smokers, smokers and patients with COPD. A small RNA sequencing (RNA-seq) approach was adapted to identify novel circulating EV miRNAs. We found that plasma-derived EVs from non-smokers, smokers and patients with COPD vary in their size, concentration, distribution and phenotypic characteristics as confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of EV surface markers. RNA-seq analysis confirmed the most abundant types of small RNAs, such as miRNAs, tRNAs, piRNAs snRNAs, snoRNAs and other biotypes in plasma-derived EVs. We mainly focused on miRNAs as novel biomarkers in smokers and patients with COPD for further analysis. Differential expression by DESeq2 identified distinct miRNA profiles (up-regulated: miR-22-3p, miR-99a-5p, miR-151a-5p, miR-320b, miR-320d; and down-regulated: miR-335-5p, miR-628-3p, miR-887-5p and miR-937-3p) in COPD versus smokers or non-smokers in a pairwise comparison. Gene set enrichment analysis (GSEA) of differentially expressed miRNAs revealed the top pathways, gene ontology and diseases associated with smokers and patients with COPD. We selectively validated miRNAs in EVs isolated from BEAS-2B cells treated with cigarette smoke extract by quantitative PCR analysis. For the first time, we report that plasma-derived EV miRNAs are novel circulating pulmonary disease biomarkers. Thus, molecular profiling of EV miRNAs has great translational potential for the development of biomarkers that may be used in the diagnosis, prognosis, and therapeutics of COPD.

细胞外囊泡（EV）在正常肺生理学中发挥着至关重要的作用，通过细胞间通讯维持气道稳态。EV 包括外泌体和微泡，其特征在于其磷脂双层。EV 已被认为是疾病的新型循环生物标志物，由不同细胞类型释放。在这项研究中，我们使用不同的 EV 分离和纯化方法来表征来自非吸烟者、吸烟者和 COPD 患者的血浆来源的 EV miRNA。采用小 RNA 测序 (RNA-seq) 方法来鉴定新型循环 EV miRNA。我们发现，来自非吸烟者、吸烟者和慢性阻塞性肺病患者的血浆来源的 EV 在大小、浓度、分布和表型特征方面有所不同，这一点已通过纳米颗粒跟踪分析、透射电子显微镜和 EV 表面标记物的免疫印迹分析证实。RNA-seq分析证实了血浆来源的EV中最丰富的小RNA类型，例如miRNA、tRNA、piRNA、snRNA、snoRNA和其他生物型。我们主要关注 miRNA 作为吸烟者和 COPD 患者的新型生物标志物进行进一步分析。DESeq2 的差异表达鉴定出不同的 miRNA 谱（上调：miR-22-3p、miR-99a-5p、miR-151a-5p、miR-320b、miR-320d；下调：miR-335-5p， miR-628-3p、miR-887-5p 和 miR-937-3p) 在 COPD 中与吸烟者或非吸烟者进行配对比较。差异表达 miRNA 的基因集富集分析 (GSEA) 揭示了与吸烟者和 COPD 患者相关的主要通路、基因本体和疾病。我们通过定量 PCR 分析选择性地验证了从用香烟烟雾提取物处理的 BEAS-2B 细胞中分离出的 EV 中的 miRNA。我们首次报告血浆来源的 EV miRNA 是新型循环肺疾病生物标志物。因此，EV miRNA 的分子分析对于开发可用于 COPD 的诊断、预后和治疗的生物标志物具有巨大的转化潜力。