State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, China.
Invest Ophthalmol Vis Sci. 2024 Jun 3;65(6):29. doi: 10.1167/iovs.65.6.29.
Ubiquitination serves as a fundamental post-translational modification in numerous cellular events. Yet, its role in regulating corneal epithelial wound healing (CEWH) remains elusive. This study endeavored to determine the function and mechanism of ubiquitination in CEWH.
Western blot and immunoprecipitation were used to discern ubiquitination alterations during CEWH in mice. Interventions, including neuronally expressed developmentally downregulated 4 (Nedd4) siRNA and proteasome/lysosome inhibitor, assessed their impact on CEWH. In vitro analyses, such as the scratch wound assay, MTS assay, and EdU staining, were conducted to gauge cell migration and proliferation in human corneal epithelial cells (HCECs). Moreover, transfection of miR-30/200 coupled with a luciferase activity assay ascertained their regulatory mechanism on Nedd4.
Global ubiquitination levels were markedly increased during the mouse CEWH. Importantly, the application of either proteasomal or lysosomal inhibitors notably impeded the healing process both in vivo and in vitro. Furthermore, Nedd4 was identified as an essential E3 ligase for CEWH. Nedd4 expression was significantly upregulated during CEWH. In vivo studies revealed that downregulation of Nedd4 substantially delayed CEWH, whereas further investigations underscored its role in regulating cell proliferation and migration, through the Stat3 pathway by targeting phosphatase and tensin homolog (PTEN). Notably, our findings pinpointed miR-30/200 family members as direct regulators of Nedd4.
Ubiquitination holds pivotal significance in orchestrating CEWH. The critical E3 ligase Nedd4, under the regulatory purview of miR-30 and miR-200, facilitates CEWH through PTEN-mediated Stat3 signaling. This revelation sheds light on a prospective therapeutic target within the realm of CEWH.
泛素化作为众多细胞事件中的一种基本翻译后修饰方式发挥作用。然而，其在调节角膜上皮伤口愈合（CEWH）中的作用仍不清楚。本研究旨在确定泛素化在 CEWH 中的功能和机制。
使用 Western blot 和免疫沉淀来辨别在小鼠 CEWH 过程中的泛素化变化。神经元表达的发育下调 4（Nedd4）siRNA 和蛋白酶体/溶酶体抑制剂的干预，评估了它们对 CEWH 的影响。划痕实验、MTS 实验和 EdU 染色等体外分析用于评估人角膜上皮细胞（HCECs）中的细胞迁移和增殖。此外，转染 miR-30/200 并结合荧光素酶活性测定，以确定它们对 Nedd4 的调节机制。
在小鼠 CEWH 过程中，泛素化水平显著增加。重要的是，蛋白酶体或溶酶体抑制剂的应用都显著阻碍了体内和体外的愈合过程。此外，Nedd4 被鉴定为 CEWH 的必需 E3 连接酶。在 CEWH 过程中，Nedd4 的表达明显上调。体内研究表明，下调 Nedd4 会显著延迟 CEWH，而进一步的研究强调了它通过靶向磷酸酶和张力蛋白同源物（PTEN）调控细胞增殖和迁移的作用。值得注意的是，我们的研究结果指出 miR-30/200 家族成员是 Nedd4 的直接调节因子。
泛素化在协调 CEWH 中具有重要意义。关键的 E3 连接酶 Nedd4 在 miR-30 和 miR-200 的调控下，通过 PTEN 介导的 Stat3 信号通路促进 CEWH。这一发现为 CEWH 领域的潜在治疗靶点提供了启示。
