Integrative Transcriptomic Analysis and Single-Cell Validation Identify a Six-Hub-Gene Signature Converging on Inflammatory Signaling in Osteoarthritis.

Osteoarthritis (OA) is a heterogeneous joint disease characterized by cartilage degeneration. The interplay between extracellular matrix (ECM) remodeling, endoplasmic reticulum (ER) stress, and inflammatory signaling in OA pathogenesis remains incompletely understood. This study aimed to identify robust diagnostic biomarkers and explore the mechanistic convergence of key genes in OA cartilage through an integrated transcriptomic framework. Three independent cartilage transcriptomic datasets (GSE285234, GSE287861, GSE289464) were integrated after ComBat batch correction. Differentially expressed genes (DEGs) were identified using limma, followed by ORA and GSEA for functional enrichment. LASSO logistic regression identified hub genes for a diagnostic model and nomogram, validated by leave-one-out cross-validation (LOOCV). Consensus clustering stratified OA samples into molecular subtypes. Single-cell RNA-sequencing (scRNA-seq) data (GSE169454, GSE220243) were used to validate cell-type-specific expression. Virtual gene knockout (scTenifoldKnk) and pathway analysis inferred downstream functional consequences. Fifty-eight DEGs (predominantly downregulated) were enriched in ECM and ER protein processing pathways. Six hub genes (EIF2S1, GANAB, STT3A, XBP1, MGP, PMP22) showed robust selection stability. The diagnostic model achieved a LOOCV AUC of 0.769, a well-calibrated nomogram, and superior net benefit. Unsupervised clustering revealed two OA subtypes with divergent unfolded protein response (UPR) and TGF-β pathway activities. scRNA-seq confirmed hub gene expression in chondrocytes and other joint microenvironment cells. Notably, virtual knockout of five hub genes convergently perturbed IL-17, NF-κB, and chemokine signaling pathways. This study identified and validated a six-gene signature reflecting ECM-ER-inflammatory crosstalk in OA cartilage. The convergent perturbation of inflammatory pathways by functionally distinct hub genes reveals a mechanistic core that may serve as a diagnostic panel and a platform for targeted therapeutic investigation in OA.