Small Extracellular Vesicle-Associated Chromosome 14 microRNA Cluster Members Reveal Distinct Molecular Responses to E-Liquid Exposure in Human Lung Cells

Background/Objectives: The adoption of electronic cigarettes and vaping devices worldwide as healthier alternatives to smoking tobacco has led to a new clinical condition termed EVALI—E-cigarette or vaping-associated lung injury. The symptoms of EVALI present large overlaps with other lung pathologies and injuries, making diagnosis, treatment, and prevention difficult. Specific biomarkers that can distinguish lung cell exposure to E-liquids, and subsequently EVALI, from other forms of toxin exposure are thus an emerging requirement. Small extracellular vesicle (sEV)-associated microRNAs represent a promising source of non-invasive molecular biomarkers of lung injury. In particular, the large, imprinted microRNA cluster members on human chromosome 14 (C14), released via sEVs, were shown to be potentially important biomarkers in lung cancer. We investigated the effects of nicotine-containing E-liquid on sEV-associated C14 miRNA profiles released from human lung adenocarcinoma (A549) and bronchial epithelial (BEAS-2B) cells. Methods: Cells were exposed to different doses of nicotine or nicotine-containing E-liquid for 72 h and sEVs were isolated using size-exclusion chromatography, characterized, and their small RNA cargo was analyzed by targeted evaluation of selected microRNAs using qRT-PCR. Results: Cellular assays reproducibly showed that nicotine containing E-liquid was significantly more toxic than pure nicotine at >100 uM for both cell lines (p < 0.001). Nanoparticle tracking analysis (NTA) confirmed the expected size distribution (<200 nm) and concentration of sEVs across all the samples while f-NTA and Western blot confirmed the presence of canonical tetraspanin surface markers. Quantitative analysis of candidate microRNAs from sEVs revealed differential expression in A549 and BEAS-2B cells after treatment, especially upon exposure to E-liquid. C14 members released from untreated A549 cells were consistently less abundant than those from untreated BEAS-2B cells. Treatment with E-liquid and nicotine reversed the pattern, showing an increase in C14MC miRNAs released via sEVs from A549 cells compared to BEAS2B cells. Conclusions: Our work is the first report to show that extracellular microRNAs secreted as sEV cargo from human lung cells can be robust molecular responses to E-liquid exposure and can lead to future non-invasive biomarkers for EVALI.