基于EYA4基因的肝细胞癌负向调控网络探索

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.

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。

我们发现EYA4在肝癌中呈超甲基化、低表达，且表达水平是术后无瘤生存期及总体生存期的独立预后因素。EYA4过表达可抑制肝癌增殖、侵袭及成瘤，提示其可能为抑癌基因。表达谱芯片显示其EYA4过表达可下调包含MYCBP在内的一系列基因的表达，但其调控MYCBP并发挥抑癌作用的机制仍不清楚。本项目主要阐明了EYA4通过调控MYCBP在肝癌发展中发挥抑癌作用的机制，并对其调控C1GALT1C1的机制进行了初步探索。通过本项目研究，(1)在人肝癌组织和细胞株中证实EYA4与MYCBP表达呈负相关：分析GEO数据库中的芯片数据资料，发现MYCBP在HCC组织中的表达显著高于正常肝组织；采用Western blot、免疫组化实验对临床肝癌标本进行检测、采用qPCR、Western blot对EYA4过表达/去表达细胞株进行检测，发现不论是在肝癌组织还是细胞中EYA4与MYCBP表达均呈负相关。(2)证实挽救实验可以恢复/抑制EYA4所调控的恶性表型：构建MYCBP表达质粒重新转染两株EYA4过表达稳转株进行挽救实验。Western Blot显示其蛋白产物的表达可显著上调恢复。而集落形成实验和CCK8增殖实验显示，MYCBP再表达可部分逆转EYA4过表达所带来的集落形成与增殖的抑制效果。细胞周期分析提示其能部分恢复EYA4过表达所带来的G2/M期停滞。在EYA4去表达稳转株中转染特异性针对MYCBP的siRNA则发挥正好相反的作用。(3)阐明了EYA4通过磷酸化酶活性去磷酸化β-Catenin的S552位点，减少β-Catenin核转位并抑制β-Catenin对MYCBP的转录活化作用。(4)生存分析提示，联合EYA4和MYCBP的表达水平能够作为肝癌的潜在的预后标志物。(5)从基因组芯片中筛选出另一个靶基因C1GALT1C1，验证其在肝癌中的表达，构建了其去表达稳转株，证实其可有效抑制肝癌细胞的恶性表型。(6)对消化系统肿瘤中表观遗传组学标志物进行鉴定、网络构建及通路富集分析。本项目对EYA4在肝癌中的抑癌作用背后的靶点、通路和机制所进行的深入探索，有望为肝癌诊疗提供新思路、新方向，具有重要的理论意义和应用价值。