microRNA and non-targeted proteome analysis of liquid biopsies from the distal lung collected by Particles in Exhaled Air (PExA) reveals presence from extracellular vesicles

BackgroundEarly detection is essential to ensure prompt treatment selection for many lung disorders, which are becoming more prevalent. Unfortunately, invasive methods are often used to examine the distal parts of the lungs for diagnosis and to establish disease biomarkers. The growing need to explore the bio-molecular mechanisms in various lung diseases highlights the importance of non-invasive methods, particularly in longitudinal studies. Thus, the use of particles in exhaled air (PExA), a non-invasive technique for sampling epithelial lining fluid from distal airways, is gaining attention. MicroRNAs (miRNAs) are crucial in modulating protein expression both intracellularly and intercellularly, often transported via extracellular vesicles. Dysregulated miRNAs have been linked to many pulmonary diseases, and their relative stability, especially when encapsulated in EVs, makes them promising biomarkers. Although underreported, exhaled miRNA identified with other non-invasive techniques shows potential as a molecular tool. Therefore, here we report the initial steps in the miRNA detection in PExA, offering opportunities to study miRNA roles in both normal and diseased states in a non-invasive manner. MethodsExhaled particles were collected from healthy subjects using the PExA 2.0 instrument following prescribed guidelines. Different samples were processed for non-targeted proteome analysis using mass spectrometry and RNA extraction followed by a miRNAseq workflow for low input starting material. The detection of some miRNAs was confirmed with the miRCURY LNA miRNA PCR assays. The quality of the whole workflow was evaluated with a RNAses activity test and with synthetic controls miRNAs (spike-in kit). ResultsProteomic analysis identified over 50 proteins across experiments and samples, with a notable enrichment of proteins associated with extracellular vesicles, including blood microparticles, and secretory granules. miRNA-seq revealed 39 mature miRNAs, the majority of which have been previously reported to be detected in the airways and respiratory disorders. Some were also reported to be secreted by primary human airway epithelial cells via extracellular vesicles. Notably, miRNA-125b and members of the let-7 family were among the most abundant. Fluorometric assays showed significant RNase activity in both PExA and other lung-related samples, such as bronchoalveolar lavage fluid, suggesting that this activity originates from the airways and is independent of the sampling techniques used. The workflow for extraction and processing of the PExA collection membrane, tested with abundant synthetic miRNAs and analyzed using the miRCURY LNA miRNA PCR assay, yielded results comparable to control samples, indicating that the membrane material does not interfere with the assay. ConclusionsUsing PExA, we identified several miRNAs that are known to be dysregulated in various pulmonary disorders. The enrichment of extracellular secretory components in the core protein list from our proteomic analysis, along with the elevated RNAse activity in the respiratory tract, suggests that the detected miRNAs may be encapsulated within extracellular vesicles. These miRNAs are particularly intriguing due to their potential role in intercellular communication during both normal and pathological lung processes. Our findings suggest that PExA holds significant potential as a non-invasive tool for studying extracellular vesicle-mediated miRNA cargo in the small airways.