DNA Methylation Regulates Corneal Epithelial Wound Healing by Targeting miR-200a and CDKN2B
DNA Methylation Regulates Corneal Epithelial Wound Healing by Targeting miR-200a and CDKN2B
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DNA 甲基化通过靶向 miR-200a 和 CDKN2B 调节角膜上皮伤口愈合
DOI：
10.1167/iovs.18-25443
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发表时间：
2019-02-01
影响因子：
4.4
通讯作者：
Yan, Dongsheng
中科院分区：
文献类型：
--
作者：
Luo, Guangying;Jing, Xia;Yan, Dongsheng
目的. DNA甲基化是一种重要的表观遗传修饰，参与各种生物学过程和疾病。角膜上皮伤口愈合（CEWH）是恢复角膜损伤后的完整性和透明度所必需的。然而，DNA甲基化在CEWH中的作用仍然难以捉摸。本文主要探讨DNA甲基化在CEWH调控中的作用及其机制。斑点印迹和整体甲基化测定确定CEWH期间的DNA甲基化水平。定量RT-PCR和Western blot分析分别检测CEWH期间DNA甲基转移酶（DNMTs）、细胞周期蛋白依赖性激酶抑制剂2B（CDKN 2B）和miR-200 a的表达。用MTS法和流式细胞仪分别检测人角膜上皮细胞（HCEC）的增殖和细胞周期。体外划痕试验评估了HCEC迁移，体内鼠角膜上皮清创模型评估了伤口愈合。采用亚硫酸氢盐测序PCR方法，检测候选基因的DNA甲基化状态。转染miR-200 a模拟物或抑制剂评估miR-200 a在HCEC中的功能。进行补救实验以阐明CEWH. RECOMPORT. CONSULTUSION期间DNMT 1与miR-200 a/CDKN 2B之间的相关性。DNMT 1和DNMT 3B表达在CEWH期间显著上调，导致显著的整体DNA超甲基化。DNMT 1下调显著延迟体内CEWH，并抑制HCEC增殖和迁移。MiR-200 a抑制HCEC迁移。此外，miR-200 a和CDKN 2B被鉴定为DNA甲基化的分子靶点，并与DNMT 1有因果关系。DNMT 1介导的DNA甲基化可通过直接靶向miR-200 a和CDKN 2B而促进CEWH的过程。这一见解精确地指出了促进CEWH的新的潜在药物靶点。
PURPOSE. DNA methylation is a key epigenetic modification involved in various biological processes and diseases. Corneal epithelial wound healing (CEWH) is essential for restoring corneal integrity and transparency after injury. However, the role of DNA methylation in CEWH remains elusive. Here, we investigate the function and underlying mechanism of DNA methylation in regulating CEWH.METHODS. Dot blots and global methylation assays determined DNA methylation levels during CEWH. Quantitative RT-PCR and Western blot analysis examined the expression of DNA methyltransferases (DNMTs), cyclin-dependent kinase inhibitor 2B (CDKN2B), and miR-200a during CEWH, respectively. MTS assays and flow cytometry were used to analyze human corneal epithelial cell (HCEC) proliferation and cell cycle, respectively. The in vitro scratch wound assay assessed HCEC migration and an in vivo murine corneal epithelial debridement model evaluated wound healing. Using bisulfite sequencing PCR, we determined the DNA methylation status of the candidate genes. Transfection of miR-200a mimic or inhibitor assessed the function of miR-200a in HCECs. Rescue experiments were performed to clarify the correlation between DNMT1 and miR-200a/CDKN2B during CEWH.RESULTS. DNMT1 and DNMT3B expression was significantly upregulated during CEWH, resulting in a significant global DNA hypermethylation. DNMT1 downregulation dramatically delayed CEWH in vivo, and suppressed HCEC proliferation and migration. MiR-200a inhibited HCEC migration. Furthermore, miR-200a and CDKN2B were identified as molecular targets of DNA methylation and as having a causal connection with DNMT1.CONCLUSIONS. DNMT1-mediated DNA hypermethylation can enhance the process of CEWH by directly targeting miR-200a and CDKN2B. This insight pinpoints novel potential drug targets for promoting CEWH.