DNMT3A-mediated epigenetic silencing of SOX17 contributes to endothelial cell migration and fibroblast activation in wound healing

BACKGROUND: Wound healing, especially impaired chronic wound healing, poses a tremendous challenge for modern medicine. Understanding the molecular mechanisms underlying wound healing is essential to the development of novel therapeutic strategies. METHODS: A wound-healing mouse model was established to analyze histopathological alterations during wound healing, and the expression of SRY-box transcription factor 17 (SOX17), DNA methyltransferase 3 alpha (DNMT3A), and a specific fibroblast marker S100 calcium-binding protein A4 (S100A4) in wound skin tissues was tested by immunofluorescence (IF) assay. Cell proliferation and migration were evaluated using 5-ethynyl-2′-deoxyuridine (EdU) and Transwell migration assays. RT-qPCR and western blotting were used to measure RNA and protein expression. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the secretion of transforming growth factor-beta (TGF-β). Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) and DNA pull-down assays were performed to confirm the interaction between DNMT3A and the CpG island of the SOX17 promoter. Promoter methylation was examined by pyrosequencing. RESULTS: SOX17 and DNMT3A expression were regularly regulated during the different phases of wound healing. SOX17 knockdown promoted HUVEC migration and the production and release of TGF-β. Through establishing an endothelial cells-fibroblasts co-culture model, we found that SOX17 knockdown in HUVECs activated HFF-1 fibroblasts, which expressed α-smooth muscle actin (α-SMA) and type I collagen (COL1). DNMT3A overexpression reduces SOX17 mRNA levels. ChIP-qPCR and DNA pull-down assays verified the interaction between DNMT3A and CpG island in the SOX17 promoter region. Pyrosequencing confirmed that DNMT3A overexpression increased the methylation level of the SOX17 promoter. CONCLUSION: DNMT3A-mediated downregulation of SOX17 facilitates wound healing by promoting endothelial cell migration and fibroblast activation.