School of Basic Medical Sciences, Nanjing Medical University, Nanjing, Jiangsu 211166, P.R. China.
Key Laboratory for Neuroregeneration, Medical College of Nantong University, Nantong, Jiangsu 226001, P.R. China.
Oncol Rep. 2022 Feb;47(2). doi: 10.3892/or.2021.8240. Epub 2021 Dec 8.
C‑terminal‑binding protein 2 (CtBP2), a transcriptional co‑repressor, plays a main role in tumorigenesis and in the development of multiple tumors. Transforming growth interacting factor (TGIF) is involved in a number of cellular signal transduction pathways and is related to tumor occurrence and development. In the present study, the proteins interacting with CtBP2 were identified and the mechanisms underlying the biological activity of CtBP2 in esophageal squamous cell carcinoma (ESCC) were investigated. The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to search for known proteins interacting with CtBP2, and co‑immunoprecipitation (Co‑IP) assay was performed to validate the interactions. Reverse transcription‑quantitative PCR (RT‑qPCR), immunohistochemistry (IHC) and western blot analysis were performed to examine the expression levels of CtBP2 and TGIF in ESCC. The correlation between CtBP2 and TGIF was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) by Pearson's correlation analysis, and the co‑localization of CtBP2 with TGIF in the ECA109 cells was identified using immunofluorescence staining. XAV939 treatment, CCK‑8, 5‑ethynyl‑2'‑deoxyuridine (EdU) staining, wound healing and Transwell assays were performed to investigate the signaling pathways involved in the biological activity of CtBP2 in ECA109 cells. According to the results obtained from STRING and Co‑IP analysis, an interaction between CtBP2 and TGIF was indicated, and these proteins were co‑localized in the nucleus. CtBP2 and TGIF mRNA and protein expression levels were robustly and simultaneously increased in both ESCC tissues and cell lines. There was a direct correlation between CtBP2 and TGIF expression levels in ESCC tissues, and both were significantly associated with metastasis and survival. The TGIF and CtBP2 expression levels were significantly increased or decreased simultaneously, in ECA109 cells transfected with LV‑CtBP2 or sh‑CtBP2, and vice versa. According to the results of CCK‑8 assay, EdU staining and Transwell assay, CtBP2 promoted the proliferation, migration and invasion of ECA109 cells through the Wnt/β‑catenin pathway. On the whole, the present study demonstrates that CtBP2 interacts with TGIF and promotes the malignant progression of ESCC through the Wnt/β‑catenin pathway.
C 端结合蛋白 2(CtBP2)作为一种转录共抑制因子,在肿瘤发生和多种肿瘤的发展中发挥重要作用。转化生长因子相互作用因子(TGIF)参与多种细胞信号转导途径,与肿瘤的发生和发展有关。在本研究中,鉴定了与 CtBP2 相互作用的蛋白,并研究了 CtBP2 在食管鳞状细胞癌(ESCC)中的生物学活性的作用机制。使用搜索工具检索相互作用基因(STRING)数据库搜索与 CtBP2 相互作用的已知蛋白,并通过共免疫沉淀(Co-IP)实验验证相互作用。采用逆转录定量 PCR(RT-qPCR)、免疫组织化学(IHC)和蛋白质印迹分析检测 ESCC 中 CtBP2 和 TGIF 的表达水平。采用 Pearson 相关性分析通过基因表达谱交互分析(GEPIA)分析 CtBP2 与 TGIF 的相关性,并通过免疫荧光染色鉴定 ECA109 细胞中 CtBP2 与 TGIF 的共定位。用 XAV939 处理、CCK-8、5-乙炔基-2'-脱氧尿苷(EdU)染色、划痕愈合和 Transwell 实验研究 CtBP2 在 ECA109 细胞中的生物学活性相关的信号通路。根据 STRING 和 Co-IP 分析的结果,提示 CtBP2 与 TGIF 之间存在相互作用,并且这些蛋白在核内共定位。CtBP2 和 TGIF 的 mRNA 和蛋白表达水平在 ESCC 组织和细胞系中均显著增加。ESCC 组织中 CtBP2 和 TGIF 表达水平之间存在直接相关性,并且均与转移和生存显著相关。在转染 LV-CtBP2 或 sh-CtBP2 的 ECA109 细胞中,TGIF 和 CtBP2 的表达水平同时显著增加或降低,反之亦然。根据 CCK-8 测定、EdU 染色和 Transwell 测定的结果,CtBP2 通过 Wnt/β-连环蛋白通路促进 ECA109 细胞的增殖、迁移和侵袭。总的来说,本研究表明 CtBP2 与 TGIF 相互作用,并通过 Wnt/β-连环蛋白通路促进 ESCC 的恶性进展。