蛋白-蛋白相互作用分析鉴定 NEClN2 为（5-乙基-2-羟基苯甲基）甲胺抑制肺癌转移的作用靶点。
Analysis of Protein-Protein Interactions Identifies NECTIN2 as a Target of (5-Ethyl-2-hydroxybenzyl) Methylamine for Inhibition of Lung Cancer Metastasis.
机构信息
Center of Excellence in Cancer Cell and Molecular Biology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
出版信息
Cancer Genomics Proteomics. 2022 Sep-Oct;19(5):624-635. doi: 10.21873/cgp.20347.
BACKGROUND/AIM: Metastasis negatively affects the survival of lung cancer patients, however, relatively few compounds have potential in metastasis suppression. This study investigated the molecular targets of N,N-bis (5-ethyl-2-hydroxybenzyl) methylamine (EMD) for metastatic inhibition.
MATERIALS AND METHODS
Proteins were analyzed by proteomic and bioinformatic analyses. Protein-protein interaction (PPI) networks were created with the Search Tool for the Retrieval of Interacting Genes. The Kyoto Encyclopedia of Genes and Genomes database and hub genes were used to determine dominant pathways. Immunofluorescence and western blot analyses validated the proteomic results and investigated signaling pathways in NCI-H23 lung cancer cells.
RESULTS
A total of 1,751 proteins were common to the control, EMD and N,N-bis(5-methoxy-2-hydroxybenzyl) methylamine (MeMD) groups; 1,980 different proteins were categorized using metastatic capacity category and analyzed for unique proteins affected by EMD. Fifteen proteins were associated with cell adhesion and six with cell migration. Nectin cell adhesion molecule 2 (NECTIN2) was expressed in the control and MeMD-treated groups but not the EMD-treated group, suggesting NECTIN2 as an EMD target. PPI network showed association of NECTIN2 with proteins regulating cancer metastasis. Kyoto Encyclopedia of Genes and Genomes pathways revealed that NECTIN2 is an upstream target of cytoskeletal regulation via SRC signaling. Western blot and immunofluorescence analyses confirmed that EMD suppressed NECTIN2, and its downstream targets, including p-SRC (Y146 and Y527) and the epithelial-to-mesenchymal transition markers tight junction protein 1, vimentin, β-catenin, snail family transcriptional repressor 1 (SNAI1), and SNAI2, while increasing E-cadherin.
CONCLUSION
EMD suppressed NECTIN2-induced activation of EMT signaling. These data support the development of EMD to prevent metastasis of lung cancer.
背景/目的：转移会对肺癌患者的生存产生负面影响，然而，具有抑制转移潜力的化合物相对较少。本研究探讨了 N,N-双（5-乙基-2-羟基苄基）甲胺（EMD）抑制转移的分子靶标。
材料和方法
通过蛋白质组学和生物信息学分析来分析蛋白质。使用 Search Tool for the Retrieval of Interacting Genes 创建蛋白质-蛋白质相互作用（PPI）网络。京都基因与基因组百科全书数据库和枢纽基因用于确定主要通路。免疫荧光和 Western blot 分析验证了蛋白质组学结果，并研究了 NCI-H23 肺癌细胞中的信号通路。
结果
控制组、EMD 组和 N,N-双（5-甲氧基-2-羟基苄基）甲胺（MeMD）组共有 1751 种蛋白质；使用转移能力类别对 1980 种不同的蛋白质进行分类，并分析受 EMD 影响的独特蛋白质。有 15 种蛋白质与细胞黏附有关，6 种与细胞迁移有关。神经细胞黏附分子 2（NECTIN2）在对照组和 MeMD 处理组中表达，但在 EMD 处理组中不表达，提示 NECTIN2 是 EMD 的靶标。PPI 网络显示 NECTIN2 与调节癌症转移的蛋白质有关。京都基因与基因组百科全书通路表明，NECTIN2 是 SRC 信号通过细胞骨架调节的上游靶标。Western blot 和免疫荧光分析证实，EMD 抑制了 NECTIN2 及其下游靶标，包括 p-SRC（Y146 和 Y527）和上皮-间充质转化标志物紧密连接蛋白 1、波形蛋白、β-连环蛋白、Snail 家族转录抑制因子 1（SNAI1）和 SNAI2，同时增加了 E-钙黏蛋白。
结论
EMD 抑制了 NECTIN2 诱导的 EMT 信号的激活。这些数据支持开发 EMD 以预防肺癌转移。
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