Electronic cigarettes (e-cigs) vaping, cigarette smoke, and waterpipe tobacco smoking are associated with various cardiopulmonary diseases. microRNAs are present in higher concentration in exosomes that play an important role in various physiological and pathological functions. We hypothesized that the non-coding RNAs transcript may serve as susceptibility to disease biomarkers by smoking and vaping. Plasma exosomes/EVs from cigarette smokers, waterpipe smokers and dual smokers (cigarette and waterpipe) were characterized for their size, morphology and TEM, Nanosight and immunoblot analysis. Exosomal RNA was used for small RNA library preparation and the library was quantified using the High Sensitivity DNA Analysis on the Agilent 2100 Bioanalyzer system and sequenced using the Illumina NextSeq 500 and were converted to fastq format for mapping genes. Enrichment of various non-coding RNAs that include microRNAs, tRNAs, piRNAs, snoRNAs, snRNAs, Mt-tRNAs, and other biotypes are shown in exosomes. A comprehensive differential expression analysis of miRNAs, tRNAs and piRNAs showed significant changes across different pairwise comparisons. The seven microRNAs that were common and differentially expressed of when all the smoking and vaping groups were compared with non-smokers (NS) are hsa-let-7a-5p, hsa-miR-21-5p, hsa-miR-29b-3p, hsa-let-7f-5p, hsa-miR-143-3p, hsa-miR-30a-5p and hsa-let-7i-5p. The e-cig vs. NS group has differentially expressed 5 microRNAs (hsa-miR-224-5p, hsa-miR-193b-3p, hsa-miR-30e-5p, hsa-miR-423-3p, hsa-miR-365a-3p, and hsa-miR-365b-3p), which are not expressed in other three groups. Gene set enrichment analysis of microRNAs showed significant changes in the top six enriched functions that consisted of biological pathway, biological process, molecular function, cellular component, site of expression and transcription factor in all the groups. Further, the pairwise comparison of tRNAs and piRNA in all these groups revealed significant changes in their expressions. Plasma exosomes of cigarette smokers, waterpipe smokers, e-cig users and dual smokers have common differential expression of microRNAs which may serve to distinguish smoking and vaping subjects from NS. Among them has-let-7a-5p has high sensitivity and specificity to distinguish NS with the rest of the users, using ROC curve analysis. These findings will pave the way for the utilizing the potential of exosomes/miRNAs as a novel theranostic agents in lung injury and disease caused by tobacco smoking and vaping.

中文翻译：

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外体微RNA是香烟，水烟烟民，电子烟使用者和双重吸烟者中的新型循环生物标记。

电子烟（e-cigs）雾化，香烟烟雾和水烟吸烟与各种心肺疾病有关。microRNA高浓度存在于外来体中，在各种生理和病理功能中起重要作用。我们假设非编码RNA转录本可能通过吸烟和吸烟而成为疾病生物标记物的易感性。香烟，水烟和双重吸烟者（香烟和水烟）的血浆外泌体/电动汽车的大小，形态和TEM，Nanosight和免疫印迹分析得到了表征。外泌体RNA用于制备小型RNA文库，并使用Agilent 2100生物分析仪系统上的高灵敏度DNA分析对该文库进行定量，并使用Illumina NextSeq 500进行测序，并转换为fastq格式以定位基因。外泌体显示了各种非编码RNA的富集，包括microRNA，tRNA，piRNA，snoRNA，snRNA，Mt-tRNA和其他生物型。对miRNA，tRNA和piRNA进行的全面差异表达分析表明，不同的成对比较之间存在显着变化。将所有吸烟和吸烟组与不吸烟者（NS）进行比较时常见且差异表达的七个microRNA是hsa-let-7a-5p，hsa-miR-21-5p，hsa-miR-29b-3p ，hsa-let-7f-5p，hsa-miR-143-3p，hsa-miR-30a-5p和hsa-let-7i-5p。电子烟vs. NS组差异表达了5种microRNA（hsa-miR-224-5p，hsa-miR-193b-3p，hsa-miR-30e-5p，hsa-miR-423-3p，hsa-miR-365a-3p和hsa -miR-365b-3p），在其他三组中未表达。microRNA的基因组富集分析显示，在所有组中，前六种富集的功能发生了显着变化，包括生物学途径，生物学过程，分子功能，细胞成分，表达位点和转录因子。此外，在所有这些组中的tRNA和piRNA的成对比较揭示了它们表达的显着变化。吸烟者，水烟吸烟者，电子烟使用者和双重吸烟者的血浆外泌体具有微小RNA的共同差异表达，这可能有助于区分吸烟和吸烟对象与NS。其中，使用ROC曲线分析，has-let-7a-5p具有较高的灵敏度和特异性，可与其他用户区分开NS。这些发现将为利用外泌体/ miRNAs作为新型治疗治疗剂在吸烟和吸烟引起的肺损伤和疾病中铺平道路。