我们发现EYA4在肝细胞癌中呈低表达，其表达水平与肿瘤大小、复发转移呈负相关；过表达EYA4可抑制肝癌细胞增殖、侵袭及成瘤，提示其为抑癌基因。表达谱分析显示过表达EYA4可显著改变一系列靶基因的表达。初步探索其中一基因RAP1A，发现EYA4是通过磷酸酶活性抑制NF-κB通路的转录活化功能而下调RAP1A表达和发挥抑癌作用的。以上前期研究提示可能存在一个由EYA4及其调控的基因和通路组成的肝癌负向调控网络，本项目拟对其进行深入探索。结合EYA4同时具备转录调控及磷酸酶活性，拟从以下4方面着手：1.mRNA及蛋白水平验证EYA4对靶基因的表达调控；2.ChIP及双荧光素酶报告系统分析EYA4转录调控机制；3.co-IP、质谱分析实验阐明EYA4去磷酸化调控机制；4.恶性表型恢复实验验证和分析靶基因对肝癌生长的作用。阐明基于EYA4的肝癌负向调控网络有望为肝癌治疗提供新靶点、新思路，意义重大。

Our previous studies revealed that EYA4 gene expression was suppressed in clinical samples of hepatocellular carcinoma (HCC) and was inversely correlated with tumor size, post-operative recurrence and survival. Stable transfection of EYA4 gene into HCC cells inhibited proliferation and invasion in vitro as well as tumor formation in vivo, suggesting that EYA4 functions as a novel tumor suppressor gene. Expression microarray analysis revealed that EYA4 overexpression significantly changed the expression pattern of a series of genes in HCC cells. A study of RAP1A gene, one of the genes that was down-regulated after EYA4 transfection, revealed that EYA4 inhibits hepatocellular carcinoma via suppressing NF-κB-dependent RAP1 transactivation through its serine/threonine-phosphatase activity. Our collective data indicated a negative regulatory network consisting EYA4, downstream genes and pathways could be responsible for the tumor suppressive effect of EYA4 in HCC. We aim at the investigation of the network and illustration of its tumor suppressive mechanisms in this project. Given the fact that EYA4 is not only a transcription regulator but also a phosphatase with specificities towards both tyrosine and serine/threonine residues, we plan to carry out the study as follows. (1). Verification the mRNA and protein expression level of target genes revealed in genome expression microarray. (2). Chromatin immuno-precipitation (ChIP) and dual luciferase reporter system are adopted to study the regulation of target gene transcription by EYA4. (3). Co-immunoprecipitation (co-IP) is performed to identify target proteins that EYA4 directly regulates through its specific phosphatase activity. Mass spectrometry technique is used to locate the residues which are responsible for EYA4 phosphatase regulation. (4). Target gene re-expression/knocking out in EYA4-stable-expressing HCC cells in vitro and in vivo to evaluated and analyze the impacts of these target genes on HCC cell proliferation, invasion and tumor formation. Through the investigation of EYA4 and its negative regulatory network in HCC, hopefully we will offer new targets and pathways for HCC management.