The Role of FTO in m6A RNA Methylation and Immune Regulation in Staphylococcus aureus Infection-Related Osteomyelitis

Sijing Liu ^1,2 Kai Li ³ Mingwu Lao ³ Changhai Long ³ Ma Biao ³ Changquan Liu ³ Haoyuan He ² Chunjiang Wang ² Wangzhu Chen ² Bin Yu ^1*

• ¹ Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou, China
• ² Maoming Hospital of Traditional Chinese Medicine, Maoming, China
• ³ Second Affiliated Hospital of Guangdong Medical University, Zhanjiang, China

Background: Regulators of n6-methyladenosine (m6A) RNA modification play important roles in many diseases; however, their involvement in Staphylococcus aureus (S. aureus)-related osteomyelitis remains inadequately explored. Therefore, this study aims to investigate the role of m6A in S. aureus-related osteomyelitis and elucidate its underlying mechanisms.Methods: We downloaded the S. aureus infection-related osteomyelitis infection dataset GSE30119 from the Gene Expression Omnibus database. Initially, we constructed a diagnostic model based on m6A genes and predicted the hub node miRNAs and transcription factors by constructing a protein-protein interaction network. Subsequently, a prognostic model was built using LASSO regression, the receiver operating characteristic curve of the model was plotted, and the predictive performance of the diagnostic model was validated. Additionally, we validated the expression of fat mass and obesity-associated protein (FTO) in S. aureus-infected Raw264.7 macrophages using qPCR and western blotting. Moreover, we conducted si-FTO experiments on mouse Raw264.7 macrophages to investigate the anti-inflammatory regulatory role of si-FTO during S. aureus infection.Results: Enrichment analyses suggested that these genes were involved in pathways related to the proliferation and oxidation of various immune cells, cellular senescence, and various tumors and immune cells, as well as cell cycle-related functions. GSEA revealed that PD-1, TH1, TH2, CTLA4, and other pathways were significantly enriched in patients with high FTO expression.GSVA indicated that the differentially enriched pathways were related to included amino acid metabolism, immunity, and infection. Correlation analysis of immune infiltration revealed that monocytes, M2 macrophages, resting mast cells, and neutrophils were present in normal and diseased samples. Differences in expression were observed between the groups. The western blotting and qPCR analyses confirmed that the protein expression of FTO was reduced in macrophages after infection with S. aureus, consistent with the observed changes in mRNA expression. Furthermore, we validated that FTO may influence the regulation of inflammation through the FoxO1/NF-kB pathway.Conclusion: The m6A RNA methylation regulator FTO may serve as a potential diagnostic marker and therapeutic target, involved in the pathogenesis of S. aureus infection-related osteomyelitis. This finding provides new insights into the relationship between FTO-mediated m6A RNA methylation and osteomyelitis.