METTL3 facilitates osteoblast differentiation and bone regeneration via m6A-dependent maturation of pri-miR-324-5p

Abstract

Background

Osteoblast differentiation is essential for fracture healing and bone regeneration. miR-324-5p has been implicated in osteoporosis, but its precise role in osteogenic differentiation remains unclear. We investigated the function and regulatory mechanisms of miR-324-5p in bone marrow mesenchymal stem cells (BMSCs).

Methods

RT-qPCR was used to assess miR-324-5p expression during osteogenic differentiation of BMSCs. ALP, Alizarin Red S (ARS), Oil Red O, and TRAP staining were performed to evaluate osteoblast, adipocyte, and osteoclast differentiation. Rat femoral fracture and calvarial bone defect models were established to assess in vivo bone regeneration. Methylated RNA immunoprecipitation (MeRIP) and luciferase reporter assays were used to investigate METTL3-mediated m6A modification of pri-miR-324-5p and its regulation of ELAVL1.

Results

miR-324-5p expression increased during osteogenic differentiation, and ALP and ARS staining confirmed enhanced osteoblast activity and mineralization following miR-324-5p overexpression. Meanwhile, Oil Red O staining showed reduced adipogenic differentiation, and TRAP staining demonstrated suppressed osteoclast formation. In vivo, miR-324-5p promoted bone healing, bone mass, and bone regeneration. Mechanistically, METTL3-mediated m6A modification facilitated pri-miR-324-5p maturation, positively regulating its expression. Additionally, miR-324-5p directly targeted ELAVL1, and ELAVL1 overexpression reversed the osteogenic effects of miR-324-5p.

Conclusion

The METTL3/miR-324-5p/ELAVL1 axis plays a crucial role in osteogenic differentiation and bone regeneration, providing new insights into m6A modification-driven osteogenesis.