Extracellular Vesicle-Associated miR-222-3p and miR-186-5p as Potential Hypoxic Markers in Canine Osteosarcoma: A Preliminary In Vitro Study

The hypoxic microenvironment plays a critical role in the progression of canine osteosarcoma (OSA) by promoting different cellular responses, including the release of extracellular vesicles (EVs). Given the clinical aggressiveness of canine OSA, the aim of this study was to evaluate the miRNAome profile in EVs released in vitro by four canine OSA cell lines under hypoxic conditions. In particular, for this study we used two commercial canine osteosarcoma cell lines (D17 and D22) and two primary osteosarcoma cell lines obtained in our laboratory (Penny and Wall). D17, D22, Penny, and Wall cell lines were cultured under normoxic and hypoxic conditions (200 µM CoCl2) for 24 h. EVs were isolated by size-exclusion chromatography and characterized by nanoparticle tracking analysis and Western blotting. miRNAs extracted from EVs were then sequenced and analyzed using bioinformatics approaches. The most representative miRNAs were identified and validated by qPCR using the miRCURY LNA miRNA PCR assay. miRNome profiling identified 233 miRNAs differentially expressed in EVs across all analyzed cell lines. Among these, 94 miRNAs were detected exclusively under hypoxic conditions. From this subset, 43 miRNAs were selected for further validation by qPCR. The qPCR results showed that miR-222-3p and miR-186-5p were significantly downregulated in the Wall cell line under hypoxia (p ≤ 0.05). TargetScan and pathway enrichment analyses demonstrated that miR-186-5p regulates target genes involved in different cellular processes. In human osteosarcoma, low serum levels of miR-222-3p are associated with poor prognosis, while miR-186-5p is recognized as a key hypoxia-responsive miRNA. Collectively, these results suggest the potential of EV-associated miRNAs as biomarkers in canine OSA and support their relevance in translational and comparative oncology.