Integration of bioinformatics and multi-layered experimental validation reveals novel functions of acetylation-related genes in intervertebral disc degeneration

•Identified 19 crucial acetylation-related DEGs in IDD through integrative bioinformatics analysis.•Validated key hub genes: IL1β, SOD2, LAMP2, and IRF1, as crucial in IDD at gene and protein levels.•Single-cell RNA analysis uncovered distinct subtypes: Neutrophils and Monocytes in IDD.•Developed a logistic regression model using hub genes for accurate IDD diagnosis.•Immune infiltration and pathway analysis reveal novel insights into IDD pathogenesis. The molecular mechanisms underlying intervertebral disc degeneration (IDD) remain poorly understood. The purpose of this work is to elucidate key molecules and investigate the roles of acetylation-related RNAs and their associated pathways in IDD. Datasets GSE70362 and GSE124272 were obtained from the Gene Expression Omnibus (GEO) and combined to investigate differentially expressed genes (DEGs) associated with acetylation in IDD patients compared to healthy controls. Critical genes were pinpointed by integrating GO, KEGG and PPI networks. Furthermore, CIBERSORTx analysis was used to investigate the differences in immune cell infiltration between different groups and the biological processes (BP), cellular components (CC) and molecular functions (MF) were calculated by GSEA and GSVA. In addition, The single-cell database GSE165722 was incorporated to validate the specific expression patterns of hub genes in cells and identify distinct cell subtypes. This provides a theoretical basis for a more in-depth understanding of the roles played by critical cell subtypes in the process of IDD. Subsequently, tissues from IVD with varying degrees of degeneration were collected to corroborate the key DEGs using western blot, RT-qPCR, and immunofluorescence staining. By integrating various datasets and references, we identified a total of 1620 acetylation-related genes. These genes were subjected to a combined analysis with the DEGs from the databases included in this study, resulting in the discovery of 358 acetylation-related differentially expressed genes (ARDEGs). A comparative analysis with differentially expressed genes obtained from three databases yielded 19 ARDEGs. The PPI network highlighted the top 10 genes (IL1B, LAMP1, PPIA, SOD2, LAMP2, FBL, MBP, SELL, IRF1 and KHDRBS1) based on their protein interaction relationships. CIBERSORTx immune infiltration analysis revealed a moderate positive correlation between the gene IL1β and Mast.cells.activated, as well as a similar correlation between the gene IRF1 and Mast.