The Role of NAT10-Mediated ac4C Modification in Osteoblast Function and Bone Formation: Insights from Integrative Bioinformatics and Experimental Validation.

Recent evidence has established a significant link between N4 acetylcytidine (ac4C) mRNA modification, mediated by N Acetyltransferase 10 (NAT10), and bone metabolism. Nonetheless, the precise role and regulatory targets of NAT10, along with its associated ac4C modification in human bone formation, remain inadequately characterized. This study employed bioinformatics analysis of transcriptomic datasets from primary osteoblasts of individuals with high versus low bone mineral density (BMD), alongside a curated set of ac4C-modified genes, to identify key differentially expressed genes (DEGs) regulated by this pathway within an osteogenic context. Overall, eleven key NAT10/ac4C-associated DEGs linked to BMD status were identified: CFD, CTSF, DCXR, FADS1, GOLIM4, IMPA2, MLEC, NCLN, NT5DC2, PTGFRN, and VASP. Notably, FADS1, NT5DC2, and PTGFRN emerged as crucial ac4C-modified genes across three machine learning models. Furthermore, the tri-gene signature (FADS1/NT5DC2/PTGFRN) showed excellent diagnostic performance in distinguishing different BMD statuses. In vitro validation using MC3T3-E1 osteoblastic cells revealed that the knockdown of NAT10 via lentiviral delivery markedly impaired cell proliferation and osteogenic differentiation. This impairment was evidenced by the results of the CCK-8 proliferation assay, alkaline phosphatase staining, and Alizarin Red staining. Additionally, qRT PCR analysis demonstrated a significant downregulation of FADS1 and NT5DC2 expression subsequent to NAT10 knockdown. These findings underscore the role of NAT10-mediated ac4C modification as a pivotal regulator of osteoblast activity and gene expression programs associated with BMD. This research offers novel insights into the regulation of bone metabolism and proposes potential diagnostic markers and therapeutic targets for osteoporosis.