Molecular Insights into the Regulatory Mechanisms Mediated by Hypoxia-Conditioned Skeletal Muscle Exosomal miRNAs.

BACKGROUND: Hypoxia alters skeletal muscle metabolism and function through complex regulatory mechanisms, including exosome-mediated microRNA (miRNA) signaling. OBJECTIVES: This study profiled exosomal miRNAs from hypoxic human skeletal muscle cells (HSMCs) to explore their roles in hypoxic adaptation. DESIGN: Human skeletal muscle cells were cultured under normoxic or hypoxic conditions, and secreted exosomes were isolated for comprehensive molecular profiling. High-throughput miRNA sequencing combined with integrative bioinformatic analyses was used to uncover hypoxia-responsive regulatory networks and key miRNA hubs involved in skeletal muscle adaptation. METHODS: HSMCs were cultured under normoxic or hypoxic conditions for 24 hours. Exosomes were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis, and immunoblotting. Exosomal miRNAs (n = 3 per group) were profiled using high-throughput sequencing, followed by differential expression, target prediction, enrichment, and network analyses. RESULTS: Isolated exosomes displayed typical morphology (mean size: 82.4 ± 3.2 nm) and expressed markers CD9, CD63, and TSG101. Seventy-four miRNAs were significantly dysregulated under hypoxia (23 upregulated, 51 downregulated; FDR < 0.05, |log2FC| ⩾ 1), including upregulated hsa-miR-210-3p and downregulated hsa-miR-486-5p, hsa-miR-127-3p, and hsa-miR-126-3p. Predicted targets (~2000 genes) included 451 genes differentially expressed in hypoxic versus normoxic skeletal muscle cells. Functional enrichment highlighted cancer-related, MAPK, PI3K-Akt, and HIF-1 signaling pathways, along with muscle differentiation processes. Network analysis identified hsa-miR-20a-5p as a central regulatory hub (46 targets), followed by hsa-miR-24-3p and hsa-miR-152-3p. hsa-miR-24-3p showed the strongest disease associations in the miRNA-disease network. CONCLUSIONS: Hypoxia induces distinct exosomal miRNA signatures in skeletal muscle, regulating genes involved in differentiation, migration, and stress response. These findings suggest that exosome-mediated miRNA signaling contributes to hypoxia-driven muscle adaptation and intercellular communication.