Abstract
OBJECTIVE: Knee osteoarthritis (KOA) is characterized by structural changes. Aging is a major risk factor for KOA. Therefore, the objective of this study was to examine the role of genes related to aging and circadian rhythms in KOA. METHODS: This study identified differentially expressed genes (DEGs) by comparing gene expression levels between normal and KOA samples from the GEO database. Subsequently, we intersected the DEGs with aging-related circadian rhythm genes to obtain a set of aging-associated circadian rhythm genes differentially expressed in KOA. Next, we conducted Mendelian randomization (MR) analysis, using the differentially expressed aging-related circadian rhythm genes in KOA as the exposure factors, their SNPs as instrumental variables, and KOA as the outcome event, to explore the causal relationship between these genes and KOA. We then performed Gene Set Enrichment Analysis (GSEA) to investigate the pathways associated with the selected biomarkers, conducted immune infiltration analysis, built a competing endogenous RNA (ceRNA) network, and performed molecular docking studies. Additionally, the findings and functional roles of the biomarkers were further validated through experiments on human cartilage tissue and cell models. RESULTS: A total of 75 differentially expressed aging-circadian rhythm related genes between the normal group and the KOA group were identified by difference analysis, primarily enriched in the circadian rhythm pathway. Two biomarkers (PFKFB4 and DDIT4) were screened by MR analysis. Then, immune infiltration analysis showed significant differences in three types of immune cells (resting dendritic cells, resting mast cells, and M2 macrophages), between the normal and KOA groups. Drug prediction and molecular docking results indicated stable binding of PFKFB4 to estradiol and bisphenol_A, while DDIT4 binds stably to nortriptyline and trimipramine. Finally, cell lines with stable expression of the biomarkers were established by lentiviral infection and resistance screening, Gene expression was significantly elevated in overexpressing cells of PFKFB4 and DDIT4 and reversed the proliferation and migration ability of cells after IL-1β treatment. CONCLUSIONS: Two diagnostic and therapeutic biomarkers associated with aging-circadian rhythm in KOA were identified. Functional analysis, molecular mechanism exploration, and experimental validation further elucidated their roles in KOA, offering novel perspectives for the prevention and treatment of the disease.