Investigation into the mechanisms of collagen deposition and extracellular matrix remodeling in keloid formation using multi-omics analysis, machine learning, and weighted gene co-expression network analysis

Background The pathological healing process of keloids is characterized by the persistent proliferation of fibroblasts and an imbalance between extracellular matrix (ECM) synthesis and degradation. Objective This study aims to elucidate the key molecular regulators and signaling pathways involved in collagen deposition and ECM remodeling, and to identify novel therapeutic targets for keloid management. Methods An integrated analysis was performed combining transcriptomic data from the Gene Expression Omnibus (GSE218007) and proteomic profiling of keloid and normal skin tissues. Key targets were selected by intersecting differentially expressed genes (DEGs) and proteins (DEPs) with fibrosis-related gene sets from the GeneCards database and further refined using a random forest (RF) machine learning algorithm. Additionally, weighted gene co-expression network analysis was constructed to identify clinically significant modules. Functional validation was conducted using quantitative real-time polymerase chain reaction analysis, Western blotting, Transwell migration assay, and wound-healing assay in keloid fibroblasts (HKF-SV40). Mechanistic studies involved chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays to investigate transcriptional regulation. Results Asporin (ASPN) was identified as a hub gene exhibiting the most pronounced upregulation in keloid fibroblasts. ASPN was associated with the transforming growth factor-beta (TGF-β) signaling pathway. ASPN knockdown significantly inhibited collagen deposition by downregulating collagen type I Alpha 1 chain (COL1A1) and collagen type III Alpha 1 chain (COL3A1), facilitated ECM remodeling by upregulating matrix metalloproteinase 1 (MMP1) and matrix metalloproteinase 3 (MMP3), and suppressed cell migration. Furthermore, ASPN silencing reduced the expression of alpha-smooth muscle actin (α-SMA) and inactivated the TGF-β/SMAD signaling pathway. Mechanistically, the transcription factor activating transcription factor 3 (ATF3) transcriptionally activated ASPN expression. Further, ATF3 knockdown inhibited collagen deposition, cell migration and the TGF-β/SMAD pathway activation, while promoting ECM remodeling; however, these effects were reversed by ASPN overexpression. Conclusion The ATF3/ASPN axis might promote collagen deposition and fibrotic progression by activating the TGF-β/SMAD signaling pathway. These findings suggest that targeting the ATF3/ASPN axis may offer a promising therapeutic strategy for keloids.