Treadmill exercise induced differential miRNA expression in mouse femoral exosomes and upregulated osteogenesis-related miRNAs

PURPOSE: Treadmill exercise promotes bone growth and remodeling in mice. Exosomes are key mediators of intercellular communication; bone or osteocyte-derived exosomes deliver miRNAs to osteoblasts to influence osteogenesis. Therefore, the study aims to profile miRNAs in femoral bone-derived exosomes of mice trained on a treadmill, and the exosomal osteogenesis-related miRNAs were identified through bioinformatics analysis and molecular biology experiments. METHODS: Mice underwent treadmill training, after which exosomes were extracted from femoral bone and characterized, and miRNA expression profiles were analyzed using RNA sequencing. Thereafter, bioinformatics analysis of these differentially expressed miRNAs in the exosomes was performed, and two osteogenesis-related candidates, miR-3960 and miR-2137, were transfected into osteoblasts separately to validate their osteogenic effects. RESULTS: Treadmill exercise markedly enhanced bone formation and improved bone microstructure. Bone-derived exosomes promoted osteogenesis, nine differentially expressed miRNAs were identified, with potential mechanistic links to bone morphometric parameters; their predicted target genes were enriched in pathways related to cellular growth, osteogenesis, axonal development, muscle tissue formation, and key signaling cascades such as PI3K/AKT and MAPK. Notably, the exosomal miR-3960 and miR-2137 were osteogenesis-related; qPCR analysis confirmed that both miRNAs were upregulated, consistent with the miRNA sequencing results. Further, transfection of miR-3960 or miR-2137 into osteoblasts enhanced osteogenesis. CONCLUSIONS: The miRNAs in exosomes from femur (almost osteocytes) of treadmill-trained mice participated not only in osteogenic differentiation (notably miR-3960 and miR-2137), but also showed potential in neural development and muscle growth. The findings offer novel insights into how running exercise benefits skeletal, muscular, and neural health.