DNMT3A 介导的 SOX17 表观遗传沉默促进伤口愈合中的内皮细胞迁移和成纤维细胞活化。
DNMT3A-mediated epigenetic silencing of SOX17 contributes to endothelial cell migration and fibroblast activation in wound healing.
机构信息
The Department of Burn, Gansu Provincial Hospital, Lanzhou, China.
Gansu University of Chinese Medicine, Lanzhou, China.
出版信息
PLoS One. 2023 Oct 19;18(10):e0292684. doi: 10.1371/journal.pone.0292684. eCollection 2023.
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.
背景
伤口愈合，尤其是慢性伤口愈合受损，对现代医学构成了巨大挑战。了解伤口愈合的分子机制对于开发新的治疗策略至关重要。
方法
建立了伤口愈合的小鼠模型，以分析伤口愈合过程中的组织病理学变化，并通过免疫荧光（IF）检测试剂盒检测伤口皮肤组织中 SOX17 盒转录因子 17（SOX17）、DNA 甲基转移酶 3α（DNMT3A）和特定成纤维细胞标志物 S100 钙结合蛋白 A4（S100A4）的表达。通过 5-乙炔基-2'-脱氧尿苷（EdU）和 Transwell 迁移实验评估细胞增殖和迁移。RT-qPCR 和 Western blot 用于测量 RNA 和蛋白质表达。酶联免疫吸附测定（ELISA）用于检测转化生长因子-β（TGF-β）的分泌。染色质免疫沉淀联合 qPCR（ChIP-qPCR）和 DNA 下拉实验用于确认 DNMT3A 与 SOX17 启动子的 CpG 岛之间的相互作用。通过焦磷酸测序检测启动子甲基化。
结果
SOX17 和 DNMT3A 的表达在伤口愈合的不同阶段受到规律调控。SOX17 敲低促进了 HUVEC 的迁移以及 TGF-β的产生和释放。通过建立内皮细胞-成纤维细胞共培养模型，我们发现 HUVEC 中的 SOX17 敲低激活了 HFF-1 成纤维细胞，使其表达α-平滑肌肌动蛋白（α-SMA）和 I 型胶原（COL1）。DNMT3A 过表达降低了 SOX17 的 mRNA 水平。ChIP-qPCR 和 DNA 下拉实验验证了 DNMT3A 与 SOX17 启动子区 CpG 岛之间的相互作用。焦磷酸测序证实，DNMT3A 过表达增加了 SOX17 启动子的甲基化水平。
结论
DNMT3A 介导的 SOX17 下调通过促进内皮细胞迁移和成纤维细胞激活促进伤口愈合。
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