Abstract: BACKGROUND: Ferroptosis is strongly associated with the occurrence and progression of osteoarthritis, but the specific characteristic genes and regulatory mechanisms are not known.
OBJECTIVE: To identify osteoarthritis ferroptosis signature genes and immune infiltration analysis using the WGCNA and various machine learning methods.
METHODS: The osteoarthritis dataset was downloaded from the GEO database and ferroptosis-related genes were obtained from the FerrDb website. R language was used to batch correct the osteoarthritis dataset, extract osteoarthritis ferroptosis genes and perform differential analysis, analyze differentially expressed genes for GO function and KEGG signaling pathway. WGCNA analysis and machine learning (random forest, LASSO regression, and SVM-RFE analysis) were also used to screen osteoarthritis ferroptosis signature genes. The in vitro cell experiments were performed to divide chondrocytes into normal and osteoarthritis model groups. The dataset and qPCR were used to verify expression and correlate immune infiltration analysis.
RESULTS AND CONCLUSION: (1) 12 548 osteoarthritis genes were obtained by batch correction and PCA analysis, while 484 ferroptosis genes were obtained, resulting in 24 differentially expressed genes of osteoarthritis ferroptosis. (2) GO analysis mainly involved biological processes such as response to oxidative stress and response to organophosphorus, cellular components such as apical and apical plasma membranes, and molecular functions such as heme binding and tetrapyrrole binding. (3) KEGG analysis exhibited that differentially expressed genes of osteoarthritis ferroptosis were related to signaling pathways such as the interleukin 17 signaling pathway and tumor necrosis factor signaling pathway. (4) After using WGCNA analysis and machine learning screening, we obtained the characteristic gene KLF2. After validation by gene microarray, we found that the gene expression of KLF2 was higher in the test group than in the control group in the meniscus (P=0.000 14). (5) In vitro chondrocyte assay showed that type II collagen and KLF2 expression was lower in the osteoarthritis group than in the control group in chondrocytes (P < 0.05), while in osteoarthritis ferroptosis, mast cells activated was closely correlated with dendritic cells (r=0.99); KLF2 was closely correlated with natural killer cells (r=-1, P=0.017) and T cells follicular helper (r=-1, P=0.017). (6) The findings indicate that using WGCNA analysis and machine learning methods confirmed that KLF2 can be a characteristic gene for osteoarthritis ferroptosis and may improve osteoarthritis ferroptosis by interfering with KLF2. 

Key words: WGCNA analysis, machine learning, osteoarthritis, ferroptosis, signature genes, immune infiltration analysis, in vitro cellular assay