Analysis and Validation of Mitophagy-Related Genes in Diabetic Foot Ulcers.

Purpose: This study aimed to identify hub genes associated with mitophagy involved in the pathogenesis and progression of diabetic foot ulcer (DFU), and to characterize their immune cell infiltration features and single-cell expression profiles. Methods: DFU-related datasets (GSE80178, GSE68183) were retrieved from the GEO database. Subsequently, differentially expressed genes (DEGs) were identified via limma analysis, followed by gene set enrichment analysis (GSEA) to assess gene function enrichment. Identified DEGs were intersected with mitophagy-related genes. Machine learning (ML) algorithms were further employed to identify hub genes. Additionally, immune cell infiltration was examined via the CIBERSORT algorithm, and the correlation between the identified genes and immune infiltration was investigated. Finally, hub genes identified were validated via the single-cell RNA sequencing dataset GSE165816, and further validated using RT-PCR and Western blot (WB) assays. Results: Two hub genes, ANO6 and ALDH2, were identified and found to be significantly downregulated in the skin tissues of patients with DFU. Receiver operating characteristic (ROC) analysis demonstrated robust diagnostic potential (ANO6, AUC = 0.833, ALDH2, AUC = 0.806). Immune cell infiltration analysis demonstrated notable differences between the DFU and normal groups in naïve B cells, monocytes, resting mast cells, γδT cells, and regulatory T cells (Tregs). The findings were further validated through single-cell RNA sequencing (scRNA-seq) analysis and experimental studies, which confirmed the downregulation of ANO6 and ALDH2 in DFU tissues. Conclusion: Two mitophagy-related hub genes, ANO6 and ALDH2, were identified and validated as being significantly downregulated in DFU. Both genes demonstrated diagnostic potential and showed an association with immune cell infiltration. These findings suggest that mitophagy dysfunction may contribute to the pathophysiology of DFU, potentially through the dysregulation of inflammatory pathways and immune responses. While the results provide valuable insights into DFU and its management, further studies with larger cohorts and deeper exploration of mechanistic links to inflammation are necessary to translate these findings into therapeutic strategies.