EYA4基因抑制肝细胞癌生长的分子机制探讨

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.

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。

本研究主要着眼于EYA4在肝癌发生发展中发挥的作用及其机制。在前期研究中，我们已证明EYA4在肝细胞癌肿瘤中表达水平与肝细胞癌患者预后呈正相关，通过在人肝细胞癌细胞系中过表达及敲低EYA4并进行细胞功能学实验，证实EYA4可抑制肿瘤细胞的增殖及侵袭，提示EYA4是一种新的抑癌基因。本课题进行了以下方面研究:（1）我们根据EYA4过表达及敲低的肝细胞癌细胞系的Affymetrix基因组表达谱芯片结果及体内体外实验，筛选出靶基因RAP1A及MYCBP。（2）通过染色质免疫共沉淀、双荧光素酶报告系统、Western blot及蛋白质免疫共沉淀等实验，显示EYA4可通过去磷酸化IκBα，抑制IκBα泛素化降解，抑制p65入核，进而抑制NF-κB信号通路，最终抑制NF-κB介导的促进RAP1A转录的功能。（3）EYA4还可通过去磷酸化p552-β-catenin，进而抑制MYCBP转录发挥抑癌作用。因此，我们认为，EYA4可通过调控NF-κB/RAP1A及β-catenin/MYCBP信号通路在肝细胞癌中发挥抑癌作用。与此同时，本课题组亦借助本项目所积累的经验，在肝内胆管细胞癌（ICC）及胰腺导管腺癌（PDAC）中探索EYA4所发挥的作用。（4）在ICC中，通过对临床样本的分析及一系列实验研究，证实ICC肿瘤中EYA4表达水平与ICC患者临床预后呈正相关，并在人ICC细胞系及裸鼠体内成瘤实验中证实EYA4可抑制ICC肿瘤细胞的增殖及侵袭。（5）在PDAC中，我们发现PDAC患者肿瘤中EYA4表达水平与PDAC患者临床预后呈正相关，并且证实EYA4通过调控β-Catenin/ID2信号通路发挥抑制PDAC的作用。综上所述，在本项目中，我们主要证实了在肝细胞癌中EYA4与患者临床预后相关，可通过调控NF-κB/RAP1A及β-catenin/MYCBP信号通路发挥其抑癌作用。同时，在肝内胆管细胞癌及胰腺导管腺癌中，我们亦证实EYA4作为抑癌基因发挥作用，为肿瘤治疗提供新靶点。