Identification of Key Regulatory Genes Involved in Pyroptosis in Osteoarthritis Through Integrated Single-Cell...

This study aimed to identify pyroptosis related genes (pyroptosis RGs) in osteoarthritis (OA) and to evaluate their potential as candidate biomarkers by integrating bulk transcriptomic analysis, machine learning, and single cell RNA sequencing.

Bulk RNA sequencing datasets (GSE55235 and GSE55457) and a single cell RNA sequencing dataset (GSE216651) related to OA were obtained from the Gene Expression Omnibus database, and gene sets associated with OA and pyroptosis were retrieved from GeneCards. Differential expression analysis and weighted gene co expression network analysis (WGCNA) of GSE55235 were used to derive differentially expressed pyroptosis related genes (DEPRGs). Candidate hub genes were screened by intersecting rankings from five cytoHubba algorithms with three supervised machine learning models (least absolute shrinkage and selection operator, support vector machine-recursive feature elimination, and random forest), and their diagnostic performance was evaluated using receiver operating characteristic curve analysis in the training and validation cohorts. The potential biological roles of the identified hub genes were further examined through functional enrichment analysis, immune cell infiltration assessment, and single-cell transcriptomic profiling.

Twenty-one DEPRGs were identified. Among these, interleukin 8 (CXCL8) and NF kappa B inhibitor alpha (NFKBIA) emerged as central hub genes with consistent diagnostic performance across cohorts. Functional enrichment analyses indicated that these genes are primarily implicated in inflammatory signaling and immune regulation pathways, including responses to tumor necrosis factor and lipopolysaccharide, chemokine and cytokine activity, and Toll like receptor, TNF, and IL 17 signaling. Single-cell transcriptomic analysis demonstrated predominant expression of CXCL8 and NFKBIA in synovial macrophages. In these cells, expression of both genes was positively correlated with pyroptosis scores, and macrophages displayed the highest pyroptosis activity among the analyzed synovial cell populations in OA.

CXCL8 and NFKBIA were identified as synovium associated pyroptosis related genes in OA with promising diagnostic performance in public datasets. These in silico findings refine current understanding of OA related inflammatory pathways and generate hypotheses for future mechanistic and experimental studies.