[Screening, functional analysis and clinical validation of differentially expressed genes in diabetic foot ulcers]

Objective: To screen the differentially expressed genes (DEGs) in diabetic foot ulcers (DFUs), and to perform functional analysis and clinical validation of them, intending to lay a theoretical foundation for epigenetic therapy of chronic refractory wounds. Methods: An observational study was conducted. The gene expression profile dataset GSE80178 of DFU patients in Gene Expression Omnibus (GEO) was selected, and the DEG between three normal skin tissue samples and six DFU tissue samples in the dataset was analyzed and screened using the GEO2R tool. For the screened DEG, ClusterProfiler, org.Hs.eg.db, GOplot, and ggplot2 in the R language packages were used for Gene Ontology (GO) enrichment analysis of biological processes, molecular functions, and cellular components, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, respectively. Protein-protein interaction (PPI) analysis was performed using STRING database to screen key genes in the DEG, and GO enrichment analysis of key genes was performed using Cytohubba plug-in in Cytoscape 3.9.1 software. DFU tissue and normal skin tissue discarded after surgery were collected respectively from 15 DFU patients (7 males and 8 females, aged 55-87 years) and 15 acute wound patients (6 males and 9 females, aged 8-52 years) who were admitted to Xiang'an Hospital of Xiamen University from September 2018 to March 2021. The mRNA and protein expressions of small proline-rich repeat protein 1A (SPRR1A) and late cornified envelope protein 3C (LCE3C) were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Data were statistically analyzed with independent sample t test. Results: Compared with normal skin tissue, 492 statistically differentially expressed DEGs were screened from DFU tissue of DFU patients (corrected P<0.05 or corrected P<0.01), including 363 up-regulated DEGs and 129 down-regulated DEGs. GO terminology analysis showed that DEGs were significantly enriched in the aspects of skin development, keratinocyte (KC) differentiation, keratinization, epidermal development, and epidermal cell differentiation, etc. (corrected P values all <0.01). KEGG pathway analysis showed that DEGs were significantly enriched in the aspects of tumor-associated microRNA, Ras related protein 1 signaling pathway, and pluripotent stem cell regulatory signaling pathway, etc. (corrected P values all <0.01). PPI analysis showed that endophial protein, SPRR1A, SPRR1B, SPRR2B, SPRR2E, SPRR2F, LCE3C, LCE3E, keratin 16 (all down-regulated DEGs), and filoprotein (up-regulated DEG) were key genes of DEGs screened from DFU tissue of DFU patients, which were significantly enriched in GO terms of keratinization, KC differentiation, epidermal cell differentiation, skin development, epidermis development, and peptide cross-linking, etc. (corrected P values all <0.01). The mRNA expressions of SPRR1A and LCE3C in DFU tissue of DFU patients were 0.588±0.082 and 0.659±0.098, respectively, and the protein expressions were 0.22±0.05 and 0.24±0.04, respectively, which were significantly lower than 1.069±0.025 and 1.053±0.044 (with t values of 20.91 and 13.66, respectively, P values all <0.01) and 0.38±0.04 and 0.45±0.05 (with t values of 9.69 and 12.46, respectively, P values all <0.01) in normal skin tissue of acute wound patients. Conclusions: Compared with normal skin tissue, there is DEG profile in DFU tissue of DFU patients, with DEGs being significantly enriched in the aspects of KC differentiation and keratin function. Key DEGs are related to the biological function of KC, and their low expressions in DFU tissue of DFU patients may impede ulcer healing.