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Background <p>This study aimed to systematically identify key genes and cell types involved in the progression and healing of diabetic foot ulcer (DFU) by integrating single-cell RNA sequencing (scRNA-seq) and bulk transcriptomics (RNA-seq and microarray) datasets analysis, providing a theoretical foundation for developing targeted therapeutic strategies.</p> Methods <p>CIBERSORT was used to identify key cell types in bulk transcriptomic datasets (GSE80178 and GSE134431), followed by the exploration of cell–cell communication networks of these key cells at the scRNA-seq level (GSE165816) using Seurat and CellChat. Limma and DESeq2 were employed to identify differentially expressed genes (DEGs) between normal and ulcer skin tissues of DFU patients. The DEGs were further intersected with wound healing genes to identify key genes. The expression levels of these genes were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC).</p> Results <p>Bulk transcriptomic deconvolution suggested altered NK cell activation-state signatures in DFU ulcer tissues compared with normal skin. Analysis of the scRNA-seq dataset identified 17 major cell types, and the cell–cell communication network demonstrated significant interactions between NK cells and M2 macrophages, M1 macrophages, lymphatic endothelial cells, fibroblasts, vascular endothelial cells, and T cells. By intersecting the 289 up-regulated and 190 down-regulated genes that exhibited consistent trends in both bulk RNA-seq datasets with 425 wound healing-related genes, 11 genes were identified. Among them, <i>ANXA1, CLDN4, HBEGF, PPARD, SPRR3, KRT6A</i>, and <i>HMOX1</i> formed an interconnected PPI network. Three putative ANXA1-interacting compounds, BIBP3226, compound 46, and RF9, were retrieved from DGIdb and further assessed by molecular docking. Keratin 6A (<i>KRT6A</i>), peroxisome proliferator activated receptor delta (<i>PPARD</i>), heparin-binding EGF like growth factor (<i>HBEGF</i>), epiregulin (<i>EREG</i>), and <i>ANXA1</i> showed significant positive correlations. RT-qPCR validated higher mRNA expressions of these genes in infected diabetic wound tissues than in control tissues. Additionally, protein expression levels of HBEGF and ANXA1 also significantly increased in the infected diabetic wound group.</p> Conclusion <p>This study identifies altered NK cell activation-state signatures and an <i>ANXA1</i>-associated wound-healing gene network involving <i>KRT6A</i>, <i>PPARD</i>, <i>HBEGF</i>, and <i>EREG</i> in DFU, providing candidate immune–repair axes for future mechanistic validation.</p>

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Integrated bulk and single-cell transcriptomic analyses identify altered NK cell activation states and an ANXA1-associated wound-healing gene network in diabetic foot ulcer

  • 期刊论文

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  • 发表日期：

    2026年7月26日

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  • Jun-Yao Yang,
  • Yu-Qing Hu,
  • Xin-Yue Chu,
  • Si-Miao Chen,
  • Zhong-Ming Wu

摘要

Background

This study aimed to systematically identify key genes and cell types involved in the progression and healing of diabetic foot ulcer (DFU) by integrating single-cell RNA sequencing (scRNA-seq) and bulk transcriptomics (RNA-seq and microarray) datasets analysis, providing a theoretical foundation for developing targeted therapeutic strategies.

Methods

CIBERSORT was used to identify key cell types in bulk transcriptomic datasets (GSE80178 and GSE134431), followed by the exploration of cell–cell communication networks of these key cells at the scRNA-seq level (GSE165816) using Seurat and CellChat. Limma and DESeq2 were employed to identify differentially expressed genes (DEGs) between normal and ulcer skin tissues of DFU patients. The DEGs were further intersected with wound healing genes to identify key genes. The expression levels of these genes were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC).

Results

Bulk transcriptomic deconvolution suggested altered NK cell activation-state signatures in DFU ulcer tissues compared with normal skin. Analysis of the scRNA-seq dataset identified 17 major cell types, and the cell–cell communication network demonstrated significant interactions between NK cells and M2 macrophages, M1 macrophages, lymphatic endothelial cells, fibroblasts, vascular endothelial cells, and T cells. By intersecting the 289 up-regulated and 190 down-regulated genes that exhibited consistent trends in both bulk RNA-seq datasets with 425 wound healing-related genes, 11 genes were identified. Among them, ANXA1, CLDN4, HBEGF, PPARD, SPRR3, KRT6A, and HMOX1 formed an interconnected PPI network. Three putative ANXA1-interacting compounds, BIBP3226, compound 46, and RF9, were retrieved from DGIdb and further assessed by molecular docking. Keratin 6A (KRT6A), peroxisome proliferator activated receptor delta (PPARD), heparin-binding EGF like growth factor (HBEGF), epiregulin (EREG), and ANXA1 showed significant positive correlations. RT-qPCR validated higher mRNA expressions of these genes in infected diabetic wound tissues than in control tissues. Additionally, protein expression levels of HBEGF and ANXA1 also significantly increased in the infected diabetic wound group.

Conclusion

This study identifies altered NK cell activation-state signatures and an ANXA1-associated wound-healing gene network involving KRT6A, PPARD, HBEGF, and EREG in DFU, providing candidate immune–repair axes for future mechanistic validation.