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.

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.

Screening and identification of potential hub genes and immune cell infiltration in the synovial tissue of rheumatoid arthritis by bioinformatic approach.

Background: Rheumatoid arthritis (RA) is an autoimmune disease that affects individuals of all ages. The basic pathological manifestations are synovial inflammation, pannus formation, and erosion of articular cartilage, bone destruction will eventually lead to joint deformities and loss of function. However, the specific molecular mechanisms of synovitis tissue in RA are still unclear. Therefore, this study aimed to screen and explore the potential hub genes and immune cell infiltration in RA. Methods: Three microarray datasets (GSE12021, GSE55457, and GSE55235), from the Gene Expression Omnibus (GEO) database, have been analyzed to explore the potential hub genes and immune cell infiltration in RA. First, the LIMMA package was used to screen the differentially expression genes (DEGs) after removing the batch effect. Then the clusterProfiler package was used to perform functional enrichment analyses. Second, through weighted coexpression network analysis (WGCNA), the key module was identified in the coexpression network of the gene set. Third, the protein-protein interaction (PPI) network was constructed through STRING website and the module analysis was performed using Cytoscape software. Fourth, the CIBERSORT and ssGSEA algorithm were used to analyze the immune status of RA and healthy synovial tissue, and the associations between immune cell infiltration and RA-related diagnostic biomarkers were evaluated. Fifth, we used the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to validate the expression levels of the hub genes, and ROC curve analysis of hub genes for discriminating between RA and healthy tissue. Finally, the gene-drug interaction network was constructed using DrugCentral database, and identification of drug molecules based on hub genes using the Drug Signature Database (DSigDB) by Enrichr. Results: A total of 679 DEGs were identified, containing 270 downregulated genes and 409 upregulated genes. DEGs were primarily enriched in immune response and chemokine signaling pathways, according to functional enrichment analysis of DEGs. WGCNA explored the co-expression network of the gene set and identified key modules, the blue module was selected as the key module associated with RA. Seven hub genes are identified when PPI network and WGCNA core modules are intersected. Immune infiltration analysis using CIBERSORT and ssGSEA algorithms revealed that multiple types of immune infiltration were found to be upregulated in RA tissue compared to normal tissue. Furthermore, the levels of 7 hub genes were closely related to the relative proportions of multiple immune cells in RA. The results of the qRT-PCR demonstrated that the relative expression levels of 6 hub genes (CD27, LCK, CD2, GZMB, IL7R, and IL2RG) were up-regulated in RA synovial tissue, compared with normal tissue. Simultaneously, ROC curves indicated that the above 6 hub genes had strong biomarker potential for RA (AUC >0.8). Conclusions: Through bioinformatics analysis and qRT-PCR experiment, our study ultimately discovered 6 hub genes (CD27, LCK, CD2, GZMB, IL7R, and IL2RG) that closely related to RA. These findings may provide valuable direction for future RA clinical diagnosis, treatment, and associated research.

Cyclic fluid shear stress promotes osteoblastic cells proliferation through ERK5 signaling pathway.

Fluid shear stress plays an important role in bone remodeling, however, the mechanism of mechanotransduction in bone tissue remains unclear. Recently, ERK5 has been found to be involved in multiple cellular processes. This study was designed to investigate the potential involvement of ERK5 in the proliferative response of osteoblastic cells to cyclic fluid shear stress. We reported here that cyclic fluid shear stress promoted ERK5 phosphorylation in MC3T3-E1 cells. Inhibition of ERK5 phosphorylation attenuated the increased expression of AP-1 and cyclin D1 and cell proliferation induced by cyclic fluid flow, but promoted p-16 expression. Further more, we found that cyclic fluid shear stress was a better stimuli for ERK5 activation and cyclin D1 expression compared with continuous fluid shear stress. Moreover, the pharmacological ERK5 inhibitor, BIX02189, which inhibited ERK5 phosphorylation in a time-dependent manner and the suppression lasted for at least 4 h. Taken together, we demonstrate that ERK5/AP-1/cyclin D1 pathway is involved in the mechanism of osteoblasts proliferation induced by cyclic fluid shear stress, which is superior in promoting cellular proliferation compared with continuous fluid shear stress.

Application of Triggered EMG in the Intraoperative Neurophysiological Monitoring of Posterior Percutaneous Endoscopic Cervical Discectomy.

OBJECTIVE: To describe the rationale and application of triggered EMG (T-EMG) in intraoperative neurophysiological monitoring, and to explore the efficacy and safety of posterior percutaneous endoscopic cervical discectomy (PPECD) in the treatment of cervical spondylotic radiculopathy (CSR) under multimodal intraoperative neurophysiological monitoring (IOM). METHODS: This study was a retrospective cohort control study. The clinical data of 74 patients with single-segment CSR from June 2015 to August 2018 were analyzed retrospectively, of whom 35 underwent IOM-assisted PPECD with triggered EMG (T-EMG group), while 39 were subjected to IOM-assisted PPECD alone (IOM group). Operation time, hospital stay, and complications were recorded for both groups. The curative effect was evaluated according to the Visual Analog Scale (VAS) of neck and arm pain, Japanese Orthopaedic Association (JOA) score, and modified MacNab scale. RESULTS: Operations were successful and all patients were followed up for at least 24 (average 31.77 ± 9.51) months with no patient lost to follow-up. No significant difference was found in preoperative baseline data between the T-EMG and the IOM group (P > 0.05). Also, no significant difference was found in the operation time between the T-EMG (108.29 ± 11.44 min) and the IOM (110.13 ± 12.70 min) (P > 0.05) group, but the difference in hospital stay (T-EMG: 5.66 ± 0.99 days; IOM: 7.10 ± 1.43 days) was statistically significant (P < 0.05). The VAS for the neck and upper limbs in the two groups at 1 month post-operation (T-EMG: 2.09 ± 1.07, 2.26 ± 0.92; IOM:2.18 ± 1.05, 2.31 ± 0.77) and the last follow-up (T-EMG: 0.83 ± 0.62, 0.86 ± 0.55; IOM: 0.90 ± 0.50, 0.87 ± 0.61) were significantly different from the preoperative scores (T-EMG: 6.14 ± 1.09, 7.17 ± 1.04; IOM: 6.18 ± 1.28, 7.15 ± 1.23) (P < 0.05). However, no significant difference was found between the two groups (P > 0.05). The 1-month postoperative JOA scores for the two groups (12.69 ± 0.76; 12.59 ± 0.82) and those at the last follow-up (14.60 ± 0.77; 14.36 ± 0.78) were significantly different from the preoperative scores (11.09 ± 0.98; 11.05 ± 0.89) (P < 0.05), but the difference between the two groups was not significant (P > 0.05). One patient in the T-EMG group developed a transient aggravation of symptoms on the first day after surgery. In the IOM group, three patients had intraoperative cerebrospinal fluid leakage, and symptoms of C5 nerve root paralysis were presented in four patients following surgery. Compared with the IOM group, the T-EMG group had fewer complications (1/35; 7/39, P < 0.05). At the last follow-up, the modified MacNab criteria were 91.43% (32/35) and 89.7% (35/39) for the T-EMG group and IOM group, respectively. CONCLUSIONS: Triggered EMG prevents the occurrence of neurological complications, which not only aids PPECD for CSR treatment in achieving satisfactory results, but also reduces average hospital stay and complication rates.