Targetome-defined miR-181c signaling from extracellular vesicles governs periodontal MSC fate via RNF150-MAP3K5

BACKGROUND: In regenerative settings, robust differentiation of human mesenchymal stromal cells (hMSCs) requires precise coupling between post-transcriptional regulation and kinase-driven pathways to achieve optimal therapeutic efficacy. miRNA-mediated regulation is widely implicated in stem cell fate control, yet its mechanistic contribution to lineage commitment remains incompletely defined. METHODS: Multi-conditioned periodontal ligament-derived hMSCs were developed as the regenerative model for alveolar bone. Extracellular vesicle (EV)-encapsulated miRNAs were delineated through microarrays. AGO2 RNA-immunoprecipitation sequencing with transcriptome profiling was integrated to establish the miRISC-associated targetome. Protein-protein interactions and signaling hierarchy were examined by Co-IP, WB, and IFC under knockdown/overexpression with pharmacological interventions. The translational efficacy of EV and miRNA was validated using a mouse ligature-induced periodontitis model, followed by µCT and histological analyses. RESULTS: EVs produced by hMSCs with higher intrinsic osteogenic capacity exhibited enhanced osteo-inductivity, traced to the enrichment of miR-181c. miR-181c was consistently upregulated during osteogenesis, with gain- and loss-of-function producing concordant effects. Mechanistically, the E3 ubiquitin ligase RNF150 emerged as the main target, whose repression reduced MAP3K5 ubiquitin-mediated proteolysis and enabled p38 activation. Local administration of EVs and miR-181c both promoted alveolar bone regeneration process in vivo. CONCLUSION: Collectively, EV-miR-181c-RNF150-MAP3K5-p38 axis was proposed linking miRNA-mediated repression to kinase activation and lineage commitment. This work provides the framework for how EV-delivered miRNAs gate hMSCs' fate decision, with significance for acellular therapeutic modalities in periodontal and skeletal tissues.