ALKBH5-mediated m6A demethylation modification of RAD51 inhibits osteogenic differentiation via promoting DNA damage in osteoporosis.

Osteoporosis (OP) is a metabolic disorder characterized by reduced bone mineral density and degeneration of bone tissue microarchitecture. Osteogenic differentiation plays a pivotal role in OP pathogenesis by facilitating new bone formation, preserving bone strength and density, and counteracting bone resorption. RNA epigenetic modifications have been increasingly implicated in multiple aspects of bone metabolism. Our previous studies revealed the regulatory role of RAD51 in OP progression. This study aimed to investigate whether RAD51 undergoes RNA methylation modification to participate in OP and to elucidate its underlying mechanisms. MC3T3-E1 cells were induced to undergo osteogenic differentiation and exposed to a simulated microgravity environment to establish an in vitro OP model. An ovariectomized (OVX) murine OP model was also established. RNA methylation level was quantified using dot blot assay. RT-qPCR was employed to analyze mRNA expression of m6A methyltransferases and demethylases. Osteogenic differentiation capacity was assessed by Alizarin Red S and alkaline phosphatase (ALP) staining. Protein expressions were evaluated by Western blot. The interaction between RAD51 and AlkB Homolog 5 (ALKBH5)/YTH domain family (YTHDF)1 was validated through RNA immunoprecipitation and dual-luciferase reporter assays. Results demonstrated that ALKBH5-mediated m6A demethylation significantly suppressed RAD51 expression in MC3T3-E1 cells. Furthermore, ALKBH5 knockdown enhanced osteoblast differentiation by alleviating DNA damage. Mechanistically, the ALKBH5/YTHDF1 m6A regulatory axis modulated RAD51 mRNA stability through m6A methylation dynamics. In vivo experiments revealed that ALKBH5 deletion mitigated bone loss and promoted osteoblastogenesis in OVX mice through inhibition of DNA damage pathways. Collectively, these findings indicated that ALKBH5-mediated m6A demethylation of RAD51 inhibited osteogenic differentiation by inducing DNA damage in OP, suggesting potential therapeutic targets for osteoporosis treatment.