关键词: 瘢痕疙瘩, 转录组, 成纤维细胞, 差异表达基因, 瘢痕疙瘩旁真皮组织

Abstract: 【Abstract】 Objective To explore the potential lesional range of keloids by analyzing the transcriptomic characteristics, and to provide a molecular basis for understanding the recurrence of keloids following surgical excision. Methods From July to December in 2022, 3 patients clinically diagnosed with keloids and treated with surgical excision at the Department of Dermatology and Venereology, Peking University First Hospital were included in the study. Samples of keloids and keloid-adjacent dermis were collected from these 3 patients, and normal dermal tissues adjacent to benign skin tumors were collected from 4 patients and served as controls. Dermal fibroblasts were obtained by primary cell culture and purification, which were then subsequently passaged to the second generation for transcriptome sequencing. Differential gene expression analysis, gene ontology （GO）-based functional analysis, and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis were performed. While analyzing differential expression genes, those with a fold change （FC） > 2 and a P value < 0.05 were defined as upregulated genes, whereas those with a FC < 0.5 and a P value < 0.05 were considered downregulated genes. The accuracy of the results was further validated by comparing them with published single-cell sequencing data on keloid and keloid-adjacent tissues （HRA000425 in GSA database） and single-cell sequencing data on the normal dermis （GSE130973 in GEO database）. Key genes in keloid-adjacent dermal fibroblasts were validated in tissue samples from this study and the literature. Results Compared with keloid-derived fibroblasts, keloid-adjacent and control dermal fibroblasts shared 63 upregulated genes enriched in biological processes including lipid transport （P = 0.038） and ion transport （P = 0.040）; compared with control dermal fibroblasts, keloid-adjacent and keloid-derived fibroblasts shared 56 upregulated genes enriched in the transforming growth factor β signaling pathway （P < 0.001）, etc. When comparing keloid-derived fibroblasts and keloid-adjacent fibroblasts with control dermal fibroblasts, 79 genes were uniquely upregulated only in keloid-adjacent fibroblasts; after filtering based on gene expression thresholds and consistency, 13 candidate genes （average expression level > 1 000 and variance of expression level within groups < 30 000） closely related to the transcriptomic characteristics of keloid-derived fibroblasts were identified, including genes inhibiting keloid formation such as SMAD6 and SMAD7, as well as those promoting keloid formation such as MSX1, SNAI1, and EDN1, which were enriched in the biological processes such as cell growth, ossification and cartilage development （all P < 0.01）. The enrichment analysis of the above-mentioned 13 genes on the ChEA3 website identified some enriched transcription factors, such as myogenic differentiation protein 1 （MYOD1） and myogenin （MYOG） （both P < 0.05）. Conclusions Compared with keloid-derived fibroblasts and control dermal fibroblasts, the transcriptomics of keloid-adjacent fibroblasts were characterized by high-level coexpression of genes involved in both the inhibition and promotion of keloid formation, which may provide a molecular explanation for the similarity in morphology between keloid-adjacent tissues and normal skin tissues as well as for the potential mechanisms underlying the high recurrence rate of keloids.

Key words: Keloid, Transcriptome, Fibroblasts, Differentially expressed genes, Keloid-adjacent dermis