KLF5转录激活ESM1促进胃癌恶性进展的作用及机制研究

KLF5-mediated transcriptional activation of ESM1 in promoting malignant progression of gastric cancer

摘要:

背景与目的 Krüppel样因子5（KLF5）和内皮细胞特异性分子1（ESM1）均与肿瘤发生发展密切相关，但KLF5是否通过转录调控ESM1促进胃癌进展尚不明确。本研究探讨KLF5调控ESM1表达及其对胃癌恶性生物学行为的影响。方法 收集30例胃癌组织及配对癌旁组织，采用qRT-PCR、Western blot及免疫组织化学检测KLF5和ESM1表达。利用UALCAN和JASPAR数据库分析KLF5与ESM1表达及潜在结合位点，通过双荧光素酶报告基因实验和染色质免疫共沉淀（ChIP-qPCR）验证KLF5对ESM1的转录调控作用。构建KLF5和ESM1敲低及过表达胃癌细胞模型，采用克隆形成、划痕实验及Transwell实验检测细胞增殖、迁移和侵袭能力；建立裸鼠皮下移植瘤模型评价其体内作用。结果 ESM1在胃癌组织及细胞中表达均显著高于正常对照（均P<0.05）。KLF5在胃癌组织中亦呈高表达。JASPAR数据库预测KLF5与ESM1启动子区域存在结合位点；双荧光素酶和ChIP-qPCR实验进一步证实KLF5可直接结合ESM1启动子并激活其转录。敲低KLF5或ESM1均可显著抑制AGS和HGC-27细胞增殖、迁移及侵袭能力（均P<0.05）。在KLF5敲低基础上过表达ESM1后，上述抑制效应被明显逆转（均P<0.05）。动物实验显示，敲低KLF5可抑制移植瘤生长，而过表达ESM1可部分恢复肿瘤生长能力。结论 KLF5通过直接转录激活ESM1促进胃癌细胞增殖、迁移、侵袭及体内成瘤。KLF5-ESM1轴可能成为胃癌诊断评估及靶向治疗的潜在分子靶点。

Abstract:

Background and Aims Krüppel-like factor 5 (KLF5) and endothelial cell-specific molecule 1 (ESM1) are closely associated with tumor progression. However, whether KLF5 promotes gastric cancer progression through transcriptional regulation of ESM1 remains unclear. This study investigated the regulatory relationship between KLF5 and ESM1 and their roles in GC progression.Methods Thirty paired gastric cancer and adjacent normal tissues were collected. The expression of KLF5 and ESM1 was determined by qRT-PCR, Western blot and immunohistochemistry. The UALCAN and JASPAR databases were used to analyze gene expression and predict potential binding sites. Dual-luciferase reporter assays and chromatin immunoprecipitation-qPCR (ChIP-qPCR) were performed to verify the transcriptional regulation of ESM1 by KLF5. Functional assays including colony formation, wound-healing and Transwell assays were conducted to evaluate cell proliferation, migration and invasion. A nude mouse xenograft model was established to assess the in vivo effects.Results ESM1 expression was significantly elevated in gastric cancer tissues and cell lines compared with corresponding controls (all P<0.05). KLF5 was also highly expressed in GC tissues. Bioinformatics analysis predicted a KLF5-binding site within the ESM1 promoter region. Dual-luciferase reporter and ChIP-qPCR assays confirmed that KLF5 directly bound to the ESM1 promoter and transcriptionally activated ESM1 expression. Knockdown of either KLF5 or ESM1 significantly inhibited the proliferation, migration and invasion of AGS and HGC-27 cells (all P<0.05). ESM1 overexpression markedly reversed the suppressive effects induced by KLF5 silencing (all P<0.05). In vivo experiments further demonstrated that KLF5 knockdown inhibited xenograft tumor growth, whereas ESM1 overexpression partially rescued tumor growth.Conclusion KLF5 promotes gastric cancer cell proliferation, migration, invasion, and tumorigenesis by directly transcriptionally activating ESM1. The KLF5-ESM1 regulatory axis may serve as a promising molecular target for the diagnosis and treatment of gastric cancer.