Conserved Hypoxia-Responsive miRNA Programs Define Adaptive and Stress-Limiting Regulatory Axes in Hepatocellular Carcinoma

Hypoxia-driven regulatory mechanisms play a critical role in tumor progression and therapeutic resistance in hepatocellular carcinoma (HCC), yet hypoxia-responsive microRNAs (HRMs) remain incompletely characterized. This study aimed to identify HRMs in canine HCC to evaluate their diagnostic potential and translational relevance to human disease. Next-generation sequencing of two canine HCC cell lines under normoxic and hypoxic conditions identified 332 and 321 differentially expressed miRNAs, respectively. Integrating these with tumor tissue data revealed 11 HRMs, featuring consistent upregulation of cfa-miR-210 and cfa-miR-34a, which was validated via RT-qPCR in hypoxic cells and clinical tissues. Both miRNAs were significantly elevated in plasma-derived extracellular vesicles (EVs), highlighting their value as promising circulating biomarkers (AUC 1.00 for miR-210; 0.98 for miR-34a). Target gene and pathway analyses identified shared regulatory nodes, including TGIF2 and SPRED1; and enrichment of MAPK, Ras, PI3K-Akt, and Rap1 signaling, broadly linked to hypoxia adaptation, cellular metabolism, and stress-response signaling. Cross-species comparison with human HCC datasets showed that, while miR-210 associates with poor prognosis in human HCC, miR-34a exhibits tumor-suppressive features. These findings define complementary HRM programs in canine HCC, reflecting conserved adaptive and stress-limiting regulatory mechanisms with potential diagnostic and translational relevance to human HCC.