■泛素化在许多细胞事件中充当基本的翻译后修饰。然而,其在调节角膜上皮伤口愈合(CEWH)中的作用仍然难以捉摸。本研究致力于确定泛素化在CEWH中的功能和机制。
■蛋白质印迹和免疫沉淀用于辨别小鼠CEWH期间的泛素化改变。干预措施,包括神经元表达的发育下调4(Nedd4)siRNA和蛋白酶体/溶酶体抑制剂,评估了它们对CEWH的影响。体外分析,比如划痕试验,MTS测定,和EdU染色,进行测量人角膜上皮细胞(HCECs)中的细胞迁移和增殖。此外,转染miR-30/200结合荧光素酶活性测定确定了它们对Nedd4的调节机制。
■在小鼠CEWH期间,全局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明显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期间Nedd4表达显著上调。体内研究表明,Nedd4的下调显著延迟了CEWH,尽管进一步的研究强调了它在调节细胞增殖和迁移中的作用,通过靶向磷酸酶和张力蛋白同源物(PTEN)的Stat3途径。值得注意的是,我们的研究结果确定miR-30/200家族成员是Nedd4的直接调节因子.
■泛素在协调CEWH中具有至关重要的意义。在miR-30和miR-200的调控权限下,关键E3连接酶Nedd4通过PTEN介导的Stat3信号传导促进CEWH。这一启示揭示了CEWH领域内的前瞻性治疗目标。
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.