Mechanistic insights into miR-584-5p-mediated Inhibition of PDLSCs osteogenic differentiation through H2AFZ upregulation and RUNX2 suppression

Periodontal ligament stem cells (PDLSCs) hold promise for bone regeneration, but their osteogenic differentiation is tightly regulated by various molecular mechanisms. MicroRNAs are key regulators of this process, and miR-584-5p has been identified as a potential modulator of osteogenesis. In this study, we investigated the role of miR-584-5p in the osteogenic differentiation of PDLSCs. Our findings show that overexpression of miR-584-5p inhibits osteogenic differentiation in vitro, as evidenced by reduced alkaline phosphatase activity, diminished mineralized nodule formation, and decreased expression of osteogenic markers, including ALPL, SP7, and RUNX2. In animal models, suppression of miR-584-5p enhances bone formation in both ectopic bone formation and rat calvarial defect models. Mechanistically, we demonstrate that miR-584-5p upregulates the histone variant H2AFZ, leading to its increased nuclear localization and binding to osteogenic gene promoters, including ALPL, SP7, and RUNX2, thereby repressing their expression. Furthermore, miR-584-5p directly targets RUNX2 mRNA, further suppressing its expression. Rescue experiments confirmed that knockdown of H2AFZ or overexpression of RUNX2 mitigates the suppressive effects of miR-584-5p on osteogenesis. Our study reveals that miR-584-5p inhibits osteogenic differentiation of PDLSCs through dual mechanisms: H2AFZ upregulation and RUNX2 suppression, offering novel insights into the epigenetic regulation of bone formation and potential therapeutic strategies for bone regeneration.