EYA4基因抑制肝细胞癌生长的分子机制探讨

项目名称： EYA4基因抑制肝细胞癌生长的分子机制探讨

项目编号： No.81472261

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 殷晓煜

作者单位： 中山大学

项目金额： 72万元

中文摘要： 我们前期研究发现肝癌组织EYA4基因呈超甲基化，其表达水平与肿瘤大小、术后复发转移负相关；转染EYA4能抑制肝癌细胞体外增殖、侵袭和体内生长，提示EYA4是一新的抑癌基因；基因组表达芯片分析示转染EYA4显著下调肝癌细胞中6个基因表达(幅度≥75%)。本项目旨在剖析EYA4调控6个目标基因的机制及各基因在EYA4抑癌功能中所起作用。(1)反相蛋白微阵验证目标基因及其相关信号通路基因的蛋白表达，染色质免疫共沉淀及双荧光素酶报告系统分析EYA4转录调控目标基因机制；(2)蛋白免疫共沉淀及GST-pulldown实验分析受EYA4磷酸酶活性直接调控的靶蛋白，质谱分析检测EYA4调控靶蛋白的具体位点，阐明EYA4去磷酸化调控的机制；(3)构建6个目标基因表达载体，体外、体内转染实验分析目标基因对肝癌生长的作用。本项目有望证明EYA4是一新抑癌基因并阐明其抑癌机制，为肝癌治疗提供新靶点，意义重大。

中文关键词： C09_肝和肝内胆管肿瘤；肝细胞癌；EYA4基因；抑癌基因

英文摘要： Our previous study revealed that EYA4 gene was hypermethylated in hepatocellular carcinoma (HCC), and its expression level was inversely related to tumor size, recurrence and survival. Stable transfection of EYA4 gene inhibited the proliferation and invasion of HCC cells in vitro as well as HCC growth in vivo, indicating that EYA4 functions as a novel tumor suppressor gene. Genome expression microarray revealed that EYA4 overexpression significantly downregulated the expression of six genes (≥75%) in HCC cells. We aim at conducting a thorough investigation of the underlying mechanisms of how EYA4 regulates these target genes and what roles these target genes play in tumor suppressive functions of EYA4 gene. (1). Reverse-phase protein microarray is adopted to verify the protein expression level of the six target genes as revealed in genome expression microarray and their relevant signaling pathway genes. Chromatin immuno-precipitation and dual luciferase reporter system are performed to investigate the molecular mechanisms of how EYA4 regulates the transcription of the target genes. (2). Co-immunoprecipitation and GST-pulldown assays are applied to identify the target proteins that EYA4 directly regulates through its specific phosphatase activity. Mass spectrometry technique is adopted to locate the residues which are responsible for EYA4 phosphatase regulation. (3).Expressing plasmids of six target genes are constructed and transfected respectively into EYA4-stable-expressing HCC cells in vitro and tumors in vivo. The impacts of these target genes on HCC cell proliferation, invasion and tumor formation are evaluated. Completion of this project is in hope of demonstrating EYA4 as a novel tumor suppressor gene and revealing the underlying mechanisms, as well as offering novel targets for HCC management.

英文关键词： C09_tumors of liver and intrahepatic bile ducts;hepatocellular carcinoma;EYA4 gene;tumor suppressor gene