Breed-by-Altitude Interaction Reveals Cardiac miRNA–mRNA Regulatory Hubs Associated with Hypoxia Adaptation in Tibetan Pigs

MicroRNAs (miRNAs) are important post-transcriptional regulators of cardiac responses to hypoxia, but how breed and altitude interact to shape cardiac miRNA expression remains unclear. We used a 2 × 2 factorial design involving Tibetan and Yorkshire pigs raised at high (3000 m) or low (100 m) altitude. Small RNA sequencing identified 1104 miRNAs, including 370 known and 734 putative novel miRNA candidates. Pairwise comparisons revealed greater breed-related divergence at high altitude and a stronger altitude-associated miRNA response in Tibetan pigs than in Yorkshire pigs. The breed-by-altitude interaction analysis identified 94 breed-by-altitude interaction-responsive miRNAs, including 25 not detected by pairwise comparisons. K-means clustering further identified a Tibetan high-altitude-enriched miRNA module whose predicted targets were associated with hypoxia-inducible factor 1 (HIF-1), mechanistic target of rapamycin (mTOR), adrenergic, calcium, and metabolic pathways. Integration with previously published cardiac messenger RNA (mRNA) differential-expression results for high-altitude Tibetan versus Yorkshire pigs, generated from the corresponding biological samples, prioritized 319 anti-correlated miRNA–mRNA pairs involving 19 miRNAs and 224 mRNAs, with ssc-miR-671-5p as the leading network hub. Quantitative real-time polymerase chain reaction (qRT-PCR), correlation analysis, and miRanda prediction prioritized three candidate regulatory pairs: ssc-miR-671-5p–ATP1B2, novel-miR-212–ADCY6, and ssc-miR-296-3p–PFKM. These findings characterize breed-dependent, altitude-associated cardiac miRNA expression patterns and prioritize candidate miRNA–mRNA relationships for future functional investigation.