House Dust Mite Induces Distinct Cellular and Extracellular Vesicle-Associated miRNA Signatures in BEAS-2B Cells: Targeted Analysis of IL-4 Responses

Airway epithelial cells actively regulate allergic asthma through allergen sensing, type 2 cytokine responses, and extracellular vesicle (EV)-mediated intercellular communication. MicroRNAs (miRNAs) are important post-transcriptional regulators of epithelial inflammation and remodeling; however, the extent to which allergen exposure differentially alters cellular and EV-associated miRNA profiles remains incompletely understood. This study characterized miRNA responses to house dust mite (HDM) exposure in BEAS-2B bronchial epithelial cells and their EV-enriched fractions and subsequently examined selected HDM-responsive miRNAs following interleukin-4 (IL-4) stimulation. BEAS-2B cells were exposed to HDM extract or IL-4 for 72 h. EV-enriched preparations were isolated from conditioned media and characterized by transmission electron microscopy, nanoparticle tracking analysis, immunoblotting of EV-associated markers, and uptake assays. Global cellular and EV-associated miRNA profiling was performed in control and HDM-treated samples using the NanoString nCounter Human v3 miRNA assay, followed by qRT-PCR validation of selected candidates. HDM-responsive miRNAs were subsequently evaluated in IL-4-treated cells and corresponding EV-associated RNA by targeted qRT-PCR. Differentially expressed miRNAs were subjected to pathway-enrichment analysis. HDM exposure induced substantial remodeling of the cellular miRNA landscape, with 39 differentially expressed miRNAs identified in BEAS-2B cells. In contrast, EV-associated RNA exhibited a smaller but distinct set of differentially expressed miRNAs, including miR-612, miR-302d-3p, miR-125b-5p, and miR-15a-5p. Notably, miR-15a-5p displayed opposite directional changes in cellular and EV-associated RNA, highlighting compartment-specific differences in miRNA abundance. qRT-PCR independently supported several EV-associated candidates, including miR-612, miR-302d-3p, miR-15a-5p, and miR-125b-5p. Pathway analyses linked HDM-responsive miRNAs to cell-cycle regulation, p53 signaling, FoxO signaling, autophagy, and inflammatory pathways. Targeted qRT-PCR analysis revealed that many selected HDM-responsive miRNAs exhibited reduced expression following IL-4 stimulation compared with their respective untreated controls, indicating different directional responses for several candidates across the two stimulation paradigms . HDM exposure is associated with distinct cellular and EV-associated miRNA responses in the BEAS-2B bronchial epithelial model. The limited overlap between cellular and EV-associated miRNA profiles indicates that specific miRNAs are present at different levels in intracellular and EV compartments. In addition, several HDM-responsive miRNAs exhibited divergent responses following IL-4 stimulation, supporting context-dependent regulation by allergen and cytokine signaling. These findings identify EV-associated miRNAs as candidates for future mechanistic studies in the context of epithelial intercellular communication and allergic airway inflammation.