Hypoxic BMSCs accelerate femur fracture healing via the METTL3/SLC2A3 m6A-glycolysis axis

Hypoxia promotes the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), thereby accelerating fracture healing. However, the underlying molecular mechanisms remain unclear. A rat fracture model was established using external force. BMSCs were isolated, identified, and used for in vitro experiments. Flow cytometry was used to assess cell surface markers CD29, CD44, and CD45. Hypoxia-related differentially expressed genes (DEGs) were identified using methylated RNA immunoprecipitation-sequencing (MeRIP-seq) (n = 3, data size: 6G). Functional enrichment analyses (gene ontology and Kyoto encyclopedia of genes and genomes) were performed on the DEGs. SLC2A3 and METTL3 were transfected using the Lipofectamine 2000 kit. Osteogenic capacity was evaluated using Alizarin Red staining, alkaline phosphatase content, glucose uptake, and ATP levels. RNA pull-down and immunoprecipitation analyses were used to verify the interaction between SLC2A3 and METTL3. Western blotting and MeRIP-qPCR were performed to assess the expression levels of these molecules. Hypoxia enhanced the osteogenic capacity of BMSCs. MeRIP-seq analysis revealed that SLC2A3 was differentially expressed in BMSCs under hypoxia treatment. SLC2A3 enhanced osteogenic capacity through the glycolytic pathway by increasing ATP levels, glucose uptake, and the expression of HK2, PKM2, and LDHA. METTL3 demonstrated significantly higher expression in the Hypoxia group than in the Ctrl group. METTL3 further enhanced osteogenesis through the glycolytic pathway by mediating m6A modification of SLC2A3. Results also revealed a binding relationship between METTL3 and SLC2A3. In vivo, SLC2A3 overexpression accelerated fracture healing. Hypoxia promotes METTL3-mediated m6A modification of SLC2A3, thereby enhancing glycolysis and osteogenesis in BMSCs. METTL3–SLC2A3 axis may serve as a therapeutic target for accelerating fracture healing.