Evaluation of small extracellular vesicular microRNAs as biomarkers for exposure of nicotine and E-liquids on human lung cells

Nicotine intake and E-Cigarette or vaping product use-associated lung injury (EVALI) can cause pulmonary disease with damaging health effects. In this study, we explored miRNAs from small extracellular vesicles (sEVs) for nicotine and E-liquid-associated biomarkers. We investigated miRNAs using NGS and further focused on a large miRNA cluster on C14q32 (C14MC), known to be dysregulated in lung-associated diseases. We asked if E-liquid exposure alters the release of these miRNAs via sEVs, compared to nicotine. We treated lung epithelial cell lines A549 and BEAS-2B with E-liquid (with and without nicotine) and commercial nicotine (100μM) for 72hrs. After treatment, sEVs were isolated using the Size Exclusion Chromatography (SEC) method, followed by total RNA isolation. Isolated sEVs were visualized using Transmission Electron Microscopy (TEM) and uptake was detected using fluorescent microscopy. Characterization of sEVs was performed using nanoparticle tracking analysis (NTA), fluorescent NTA (fNTA) and western blot. MiRNA expression profile for miRNAs from C14MC was measured by qPCR using the 2^ΔΔCt method. Massively parallel sequencing of small RNA was performed to identify sEV miRNA as novel biomarker. TEM, NTA, fNTA and western blot – all confirmed the successful isolation and processing of small EVs. From C14MC, differential expression of sEV miRNA was identified in A549 and BEAS-2B when treated with nicotine, E-liquid (strawberry) and E-liquid (apple). As a representative example, miR-382, which was downregulated with nicotine and apple flavored E-liquid but upregulated with strawberry flavored E-liquid. The downregulation of miR-382 was also confirmed by next-generation sequencing approach. From C14MC, other notable miRNAs that showed the potential as biomarkers for E-liquid exposure are, miR-410, miR-541, miR-758 and miR-889. In addition to miR-382, the NGS approach has also identified miR-184, miR-6834 and miR-10b as potential biomarkers which are not within the C14MC region. Thus, these candidates can potentially serve as non-invasive biomarkers to identify the effect of nicotine and E-liquid exposure on human lung cells. Keywords: sEVs, E-liquid, EVALI, Nicotine, miRNA, biomarker