Unraveling Key m6A Modification Regulators Signatures in Postmenopausal Osteoporosis through Bioinformatics and Experimental Verification.

OBJECTIVE: Bone marrow mesenchymal stem cells (BMSCs) show significant potential for osteogenic differentiation. However, the underlying mechanisms of osteogenic capability in osteoporosis-derived BMSCs (OP-BMSCs) remain unclear. This study aims to explore the impact of YTHDF3 (YTH N6-methyladenosine RNA binding protein 3) on the osteogenic traits of OP-BMSCs and identify potential therapeutic targets to boost their bone formation ability. METHODS: We examined microarray datasets (GSE35956 and GSE35958) from the Gene Expression Omnibus (GEO) to identify potential m6A regulators in osteoporosis (OP). Employing differential, protein interaction, and machine learning analyses, we pinpointed critical hub genes linked to OP. We further probed the relationship between these genes and OP using single-cell analysis, immune infiltration assessment, and Mendelian randomization. Our in vivo and in vitro experiments validated the expression and functionality of the key hub gene. RESULTS: Differential analysis revealed seven key hub genes related to OP, with YTHDF3 as a central player, supported by protein interaction analysis and machine learning methodologies. Subsequent single-cell, immune infiltration, and Mendelian randomization studies consistently validated YTHDF3's significant link to osteoporosis. YTHDF3 levels are significantly reduced in femoral head tissue from postmenopausal osteoporosis (PMOP) patients and femoral bone tissue from PMOP mice. Additionally, silencing YTHDF3 in OP-BMSCs substantially impedes their proliferation and differentiation. CONCLUSION: YTHDF3 may be implicated in the pathogenesis of OP by regulating the proliferation and osteogenic differentiation of OP-BMSCs.

METTL3-mediated m6A modification of SOX4 regulates osteoblast proliferation and differentiation via YTHDF3 recognition.

N6-methyladenosine (m6A), the most prevalent internal modification in mRNA, is related to the pathogenesis of osteoporosis (OP). Although methyltransferase Like-3 (METTL3), an m6A transferase, has been shown to mitigate OP progression, the mechanisms of METTL3-mediated m6A modification in osteoblast function remain unclear. Here, fluid shear stress (FSS) induced osteoblast proliferation and differentiation, resulting in elevated levels of METTL3 expression and m6A modification. Through Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and Transcriptomic RNA Sequencing (RNA-seq), SRY (Sex Determining Region Y)-box 4 (SOX4) was screened as a target of METTL3, whose m6A-modified coding sequence (CDS) regions exhibited binding affinity towards METTL3. Further functional experiments demonstrated that knockdown of METTL3 and SOX4 hampered osteogenesis, and METTL3 knockdown compromised SOX4 mRNA stability. Via RNA immunoprecipitation (RIP) assays, we further confirmed the direct interaction between METTL3 and SOX4. YTH N6-Methyladenosine RNA Binding Protein 3 (YTHDF3) was identified as the m6A reader responsible for modulating SOX4 mRNA and protein levels by affecting its degradation. Furthermore, in vivo experiments demonstrated that bone loss in an ovariectomized (OVX) mouse model was reversed through the overexpression of SOX4 mediated by adeno-associated virus serotype 2 (AAV2). In conclusion, our research demonstrates that METTL3-mediated m6A modification of SOX4 plays a crucial role in regulating osteoblast proliferation and differentiation through its recognition by YTHDF3. Our research confirms METTL3-m6A-SOX4-YTHDF3 as an essential axis and potential mechanism in OP.

Marrow stromal stem cell autologous transplantation in denervated fracture healing: an experimental study in rats.

OBJECTIVE: To investigate the influence of bone marrow stromal stem cell (BMSCs) transplantation on healing of fractures combined with central nerve injuries in rats. METHODS: Forty-eight healthy adult SD male rats were randomly divided into the following three groups (16 rats in each group): group A, simple (left) tibial fracture; group B, tibial fracture combined with T10 spinal cord transection (SCT); group C, tibial fracture combined with T10 SCT and BMSCs transplantation. The tibial fractures were stabilized with modular intramedullary nails and all operated hind limbs were further immobilized in plaster casts to prevent unequal load bearing. BMSCs were labeled with bromodeoxyuridine and implanted into the fractures of C group rats 2 days after creation of the model. The animals in B and C groups were evaluated by postoperative Tarlov scores. The fractured tibiae were evaluated separately radiographically (X-ray and CT) and immunohistochemically 1, 2, 3 and 4 weeks after injury to assess fracture healing. In addition, the wet weights of the left tibias were measured. RESULTS: All Tarlov score of the B and C group animals reached the requirements of the experiment. One, 2 and 3 weeks after surgery, the tibial callus widths in B and C group animals were significantly greater than those of group A rats (P < 0.05). At 4 weeks the tibial callus width in group C animals had decreased, but still differed significantly from that in group A rats (P < 0.05). One, 2, 3 and 4 weeks after surgery, the wet weights of B and C group tibias were significantly greater than those of group A (P < 0.05). Hematoxylin-eosin-stained sections showed bony union and increased bone trabecula in B and C groups and areas with particles positive for alkaline phosphatase staining were more abundant in groups B and C, especially in group C. CONCLUSION: Neural regulation plays an important role in fracture healing. Treatment with BMSCs has a positive effect on defective callus in rats that have been subjected to SCT.