Deciphering and verifying cuproptosis-associated hub genes in diabetic foot ulcer by combining single-cell and bulk RNA-sequencing

Prior studies have indicated elevated plasma copper levels in patients with diabetes compared with healthy controls. However, cuproptosis remains unexplored in diabetic foot ulcers (DFUs). DFU-associated datasets (GSE165816, GSE80178, and GSE134431) were obtained from the Gene Expression Omnibus (GEO) database. Cuproptosis-associated genes (CRGs) were identified by combining single-cell and bulk RNA-sequencing data. Differentiation, enrichment, network, pseudotime, immune infiltration, cellular communication, drug sensitivity, and molecular docking analyses were performed. The CRGs were verified in a Wistar DFUs rat model. Four hub genes were obtained (DCN, IGF1, CXCL12, and CXCL8). Enrichment analysis indicated that these genes were involved primarily in cytokine storms. Moreover, network analysis revealed the relationships among competing endogenous RNAs, transcription factors, single nucleotide polymorphisms, and hub genes. In addition, pseudotime analysis revealed greater numbers of plasma cells, naive B cells, and CD4 ⁺ T cells in DFUs than controls. Furthermore, immune infiltration analysis indicated immune cells dysregulation in DFUs, characterized by lower numbers of activated mast cells, activated NK cells, and M1 macrophages than those in controls. In addition, cellular communication analysis revealed that mesenchymal stem cells frequently interacted with fibroblasts, keratinocytes, endothelial cells, and T cells. The nomogram indicated that four hub genes were included for diagnosis of DFUs and the DFU risk was approximately 0.86. Finally, drug sensitivity analysis and molecular docking demonstrated that sirolimus was an effective drug for DFU treatment. Together, our findings link IGF1 and CXCL12 to cuproptosis, thus providing novel insights for DFU diagnosis and treatment.