Senescence-Associated Checkpoint Gene Dysregulation in Established Osteoarthritis: Integrated Transcriptomic Analysis and In Vitro Evaluation of CDK6 and WEE1.

Background/Objectives: Osteoarthritis (OA) is a whole-joint disease, and cellular senescence is one of several processes associated with cartilage degeneration. This study aimed to identify senescence-associated differentially expressed genes in established OA and to examine checkpoint-related candidates without assuming a causal checkpoint-imbalance mechanism. Methods: Five Gene Expression Omnibus (GEO) datasets spanning articular-cartilage tissue and primary cartilage-derived chondrocytes (GSE57218, GSE117999, GSE114007, GSE246425, and GSE169077) were integrated as a training cohort; the meniscus dataset GSE98918 was reserved as an independent cross-tissue validation cohort. OA-associated differentially expressed genes (DEGs) were intersected with CELLAGE genes. Enrichment, protein-protein interaction, transcription-factor, competing endogenous RNA, drug-enrichment, and molecular-docking analyses were performed. CDK6 and WEE1 expression was evaluated in IL-1β-treated human C28/I2 chondrocytes by RT-qPCR and representative Western blotting. Results: Forty-one senescence-associated DEGs were identified, and seven network-central genes (CDKN1A, CDK6, WEE1, NFKB2, ID1, RBL2, and IGFBP7) were prioritized. CDKN1A, CDK6, and WEE1 were reduced in OA-associated meniscal samples in GSE98918; within-dataset ROC analyses yielded AUCs of 0.931, 0.882, and 0.792, respectively. In IL-1β-treated C28/I2 cells, WEE1 mRNA decreased whereas CDK6 mRNA increased; representative immunoblots showed concordant qualitative trends. Berberine- and folic acid-related docking findings were computational only. Conclusions: Checkpoint-related gene expression is associated with senescence-linked transcriptomic changes in established OA. The discordant CDK6 results between clinical tissue datasets and an acute inflammatory cell model do not establish stage-dependent regulation. The present data also do not demonstrate p53-mediated CDKN1A activation, checkpoint failure, cell-cycle arrest, or cellular senescence; these hypotheses require dedicated functional experiments.