Eps15同源结构域包含蛋白2通过调控Cyclin D1-CDK4-pRb信号轴影响食管鳞癌细胞的增殖

EHD2 Affects the Proliferation of Esophageal Squamous Cell Carcinoma by Regulating the Cyclin D1-CDK4-pRb Signaling Axis

微丝结合蛋白是微丝细胞骨架的重要组成成分,它们通过促进微丝的聚合和解聚来影响微丝的动力学。大量研究已经表明,微丝和微丝结合蛋白参与细胞癌变的所有阶段。我们通过对食管癌蛋白质组数据挖掘结果显示,微丝结合蛋白Eps15同源结构域包含蛋白2(EHD2)在食管癌组织中低表达,且EHD2低表达的食管癌患者预后不良。以往的研究已经证明,EHD2参与调控糖代谢、自噬和肿瘤迁移。然而,EHD2在食管癌进展中的作用和机制仍不清楚。本研究旨在探究EHD2在食管鳞癌细胞中的影响及其作用机制。免疫荧光和细胞组分分离结果显示,EHD2不仅定位于细胞膜和细胞质,还存在于细胞核中。使用克隆形成实验、EdU细胞增殖实验和细胞流式术检测EHD2对食管鳞癌细胞增殖能力的影响。结果显示,过表达EHD2和EHD2-3×NLS(核定位信号)抑制食管鳞癌细胞增殖和细胞周期G_(1)/S转换;同时,双荧光素报告基因结果显示,过表达EHD2和EHD2-3×NLS抑制Wnt信号通路活性。而siRNA敲降则获得相反的结果。免疫共沉淀和Duolink-PLA实验证明,EHD2与Wnt信号通路关键分子β-连环蛋白(β-catenin)和T细胞因子3(T-cell factor 3,TCF3)相互作用。蛋白质印迹和荧光定量PCR结果证实,过表达EHD2和EHD2-3×NLS抑制TCF3下游与增殖和细胞周期相关的靶基因的转录,以及细胞周期蛋白D1(cyclin D1)、细胞周期蛋白激酶4(CDK4)和pRb的蛋白质表达。以上结果表明,核EHD2与β-catenin和TCF3复合体相互作用,通过Cyclin D1-CDK4-pRb信号轴来调控食管鳞癌细胞的增殖和细胞周期进程。

Actin-binding proteins(ABPs)are important components of the F-actin cytoskeleton and affect the dynamics of F-actin by promoting the polymerization and depolymerization of actin.Numerous studies have shown that F-actin and actin-binding proteins are involved in all stages of carcinogenesis.Our analysis of esophageal carcinoma proteomic data showed that the actin-binding protein EHD2(Eps15 homology domain-containing protein 2)is expressed at low levels in esophageal squamous cell carcinoma tissues and patients with lower EHD2 expression had poorer prognosis.Previous studies have revealed that EHD2 is involved in the regulation of glucose metabolism,autophagy and tumor cell migration.However,the role and mechanism of EHD2 in esophageal squamous cell carcinoma progression remain unclear.This study aimed to explore the effect of EHD2 on the proliferation of esophageal squamous cell carcinoma.Immunofluorescence and cell fractionation analysis showed that EHD2 was not only localized in the cell membrane and cytoplasm,but also in the nucleus.Colony formation,EdU labeling and flow cytometry were used to determine the effect of EHD2 on the proliferation of esophageal squamous cell carcinoma.The results showed that overexpression of EHD2 and EHD2-3×NLS(nuclear localization signal)inhibited proliferation,cell cycle G_(1)/S transition,and Wnt signaling pathway activity in esophageal squamous cell carcinoma.Dual-luciferase reporter gene assay revealed that overexpression of EHD2 and EHD2-3×NLS inhibited Wnt pathway activity.While siRNA-mediated knockdown of EHD2 displayed the opposite results.Co-immunoprecipitation(Co-IP)and Duolink-PLA experiments demonstrated that EHD2 interacted withβ-catenin and TCF3,the key molecules of the Wnt signaling pathway.Western blotting and quantitative real-time PCR(qRT-PCR)results confirmed that EHD2 inhibited the transcription of TCF3 downstream target genes associated with proliferation and cell cycle,as well as the protein expression of cyclin D1,CDK4 and pRb.These results suggest that EHD2 interacts withβ-catenin and TCF3 after being transported to the nucleus,and regulates the proliferation and cell cycle progression of esophageal squamous cell carcinoma through the cyclin D1-CDK4-pRb signaling axis.