AZGP1基因与结直肠癌肝转移相关机制研究

In order to understand the molecular basis of liver metastasis(LM) of colorectal cancer(CRC),our research group analyzed the tissue from colorectal cancer using whole human gene chip. All tissues were divided into three groups: CRC without LM, CRC with synchronous LM and CRC with metachronous LM according to our own follow-up database including more than 2000 cases and the time span was longer than 10 years. We found some new genes which could be one of the important role in liver metastasis of colorectal cancer. AZGP1 was one of these genes, which was found 2.36 fold in synchronous LM and 1.77 fold in metachronous LM than those CRC without LM. We want to do some works confirming the molecular mechanism of AZGP1 in liver metastasis of CRC: 1) Clinical veification: RT-PCR and immunohistochemistry to verify the expression of AZGP1 and liver metastasis of CRC；2）To build the letivirus vectors, we would study the changes of proteins in CRC cells when we regulate the expression of AZGP1 in cell and aninmal modles. 3) We use coImmunoprecipitation to study the changes of expression of AZGP1 and its proteins, also we would focus on the changes of CRC cell behaviours. 4) We use Yeast TwoHybrid System to study the changes of expression of AZGP1 and its proteins, also we would focus on the changes of CRC cell behaviours. Through all these studies, we want to verify the molecular basis of AZGP1 in the liver metastasis of colorectal cancers.

申请者所在课题组通过基因芯片和real-time PCR验证，确认了一系列与大肠癌转移相关的特异性功能分子。由此关注到一个尚未在肿瘤中存在报道的基因- - AZGP1在同时性肝转移组较无肝转移组升高2.36倍，而在异时性肝转移组升高1.77倍，提示其可能与肝转移相关。在此基础上，本项目拟通过：1）大规模组织标本中验证AZGP1与结直肠癌肝转移的关系；2）构建过表达、RNAi和功能回复的慢病毒载体，上调和下调大肠癌细胞中AZGP1的表达，观察AZGP1表达下调后大肠癌细胞形态和侵袭转移能力的变化，并在动物模型上验证；3）应用免疫共沉淀和酵母双杂交两种不同的筛选方法，探寻与AZGP1发生相互作用的蛋白分子，通过体外和体内实验验证AZGP1与候选蛋白的相互作用并明确其作用方式，研究AZGP1在细胞信号通路中的定位，探索AZGP1在肿瘤转移发生途经中可能的定位和分子机制。

肠癌肝转移是影响肠癌患者生存的主要因素，前期通过基因芯片筛选发现AZGP1在肠癌肝转移组高表达，并在组织中得到验证。基于此，本研究进一步展开临床分析和分子生物学的研究。结果显示，通过扩大临床样本，免疫组化实验表明，AZGP1在同时性肝转移、异时性肝转移的患者中较无肝转移患者高表达。结合临床病理数据显示AZGP1高表达影响临床预后以及患者的总体生存，此外AZGP1高表达是肠癌肝转移的危险因素。体外细胞实验则显示，沉默AZGP1后肠癌细胞的增殖能力、转移侵袭能力均下降，动物实验显示，裸鼠成瘤能力以及肝转移能力均下降，提示AZGP1发挥重要的促肿瘤作用。为了进一步探索其分子机制，通过基因芯片结合通路分析以及Western Blot等的验证，发现adhesion通路可能促进肿瘤转移。同时，免疫共沉淀实验寻到到AZGP1的结合蛋白FLNA，该结合蛋白参与调控AZGP1的促转移作用，且与其亚细胞定位相关。本研究初步探明AZGP1通过介导FLNA以及 focal adhesion pathway 进而调控肠癌细胞的转移，提示其可能是肠癌肝转移的一个重要的分子标志物。