m(6)A-mediated stabilization of Bhlhe22 suppresses osteogenesis in osteoporosis by activating the PI3K-AKT-GSK3β signaling axis.

BACKGROUND: Impaired osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) contributes to osteoporosis (OP) pathogenesis. While RNA modifications such as N6-methyladenosine (m(6)A) regulate stem cell differentiation, their role in OP remains unclear. Transcriptomic analysis revealed upregulated basic helix-loop-helix family member e22 (Bhlhe22) in OP-BMSCs. This study aimed to investigate whether dysregulated m(6)A modification contributes to OP by upregulating Bhlhe22, thereby impairing the osteogenic differentiation of BMSCs. METHODS: Female Sprague-Dawley rats were subjected to ovariectomy (OVX) to establish an OP model. BMSCs were isolated, characterized by flow cytometry and trilineage differentiation, and subjected to osteogenic induction. Bhlhe22 expression was modulated via lentiviral overexpression, siRNA-mediated knockdown, and adeno-associated virus (AAV)-mediated shRNA knockdown in vivo. Gene expression was assessed by immunofluorescence, quantitative PCR (qPCR), western blotting, and RNA sequencing. Osteogenic differentiation was evaluated via alkaline phosphatase (ALP), alizarin red S (ARS) staining, and osteogenic marker expression. Micro-computed tomography (micro-CT) and histology were used to assess bone structure. Mechanisms were investigated via PI3K-Akt pathway activation (recilisib), chromatin immunoprecipitation sequencing (ChIP-seq), ChIP-qPCR, dot blot, methylated RNA immunoprecipitation (MeRIP)-qPCR, luciferase reporter assays, and site-directed mutagenesis. RESULTS: Global m(6)A levels were dysregulated in OP-BMSCs. Bhlhe22 mRNA and protein levels were significantly elevated in OP-BMSC and OVX rat femurs. Bhlhe22 knockdown restored the osteogenic potential in OP-BMSCs and increased the bone volume/trabecular thickness in OVX rats, whereas Bhlhe22 overexpression inhibited osteogenesis. BHLHE22 repressed osteogenesis by directly transactivating phosphoinositide-3-kinase regulatory subunit 3 (Pik3r3), activating the PI3K-AKT-GSK3β pathway. Crucially, m(6)A modification at position 2023 stabilized Bhlhe22 mRNA, increasing its expression. Mutation of this m(6)A site reduced the inhibitory effect of Bhlhe22. CONCLUSIONS: m(6)A modification stabilizes Bhlhe22 mRNA, leading to its overexpression in OP. Elevated BHLHE22 protein suppresses BMSC osteogenesis by transcriptionally activating Pik3r3 and the PI3K-AKT-GSK3β signaling axis. Targeting the m(6)A-Bhlhe22-PI3K axis represents a promising therapeutic strategy for osteoporosis.