胃癌5p15.3高频杂合性缺失位点内NKD2抑癌基因的功能分析

Screening loss of heterozygosity (LOH) in chromosome is an effective means of finding tumor suppressor genes. 5p15.3, a target region with high frequency of LOH is originally found in the analysis of gastric cancer by our group. Under the supporting of National Natural Science Foundation of China, we focused on candidate gene IRX1 and performed a series of functional studies. We firstly characterized the tumor suppressor functions of IRX1, which have been approved as important evidence of tumor suppressor gene by international peers. Our previous results have been inputted into NCBI database entries. Currently, thousands of human genes are recorded in database. However, the exact biological function of these genes is unclear yet, pending in-depth study. NKD2 is such a gene, located in 5p15.3 LOH locus. NKD2 is originally considered a negative regulator of Wnt signaling pathway. But recent research suggests that it may also involve in interaction with other important functional protein. Up-to-date, it lacks of systematic research for NKD2 molecular mechanism in gastric cancer. This study will focus on NKD2 gene and its relationship with gastric cancer. We will analysis the genomic alteration, mRNA transcription and protein expression of NKD2 gene. We also evaluate the biological effects based on NKD2 gene transfection in vitro. The tumorigenesis and invasive ability will be studied by nude mice experiments. The purpose of the project is to clarify the exact mechanism of NKD2 on regulating carcinogenesis and development of gastric cancer, and develop novel molecular targets for diagnosis and treatment of gastric cancer.

染色体杂合性缺失（LOH）筛查是寻找抑癌基因的一种有效手段，5p15.3是本项目组前期在胃癌研究中原创性发现的高频LOH位点。我们已就该位点的候选抑癌基因IRX1进行了一系列功能分析，明确了该基因的抑癌功能特性，研究成果获国际同行认可，并作为重要证据录入NCBI基因数据库。当前，基因数据库内登录有数万条人类基因，但相当一部分基因的确切生物学功能尚不清楚，5p15.3位点内的另一候选抑癌基因NKD2便属此类。NKD2在克隆初期被认为是Wnt信号通路的负性调控因子，但新近有研究提示其还参与其他重要功能蛋白的调控。NKD2在胃癌发生发展中的分子机制尚缺乏系统性研究。本研究从DNA水平、mRNA转录、蛋白表达、NKD2的基因过表达及转染细胞的裸鼠成瘤性与转移能力等多角度探讨NKD2基因与胃癌的关系，最终探明NKD2在胃癌发生发展中的确切作用机制，为开发胃癌诊治中的新型分子靶点提供源头数据。

NKD2基因是本课题组前期在胃癌抑癌基因筛查研究中，采用LOH方法在5号染色体5p15.3位点发现高频杂合性缺失区域内基因，该缺失区的IRX1基因已经经本课题组的系列研究确认为胃癌抑癌基因。鉴于NKD2是位于5p15.3位点的另外一个基因，该位点的高频LOH提示为潜在抑癌基因，为了明确NKD2在胃癌发生发展中是否具有抑癌基因作用，本课题拟通过对胃癌细胞株以及胃癌组织中的NKD2基因表达，基因编码区突变分析、启动子甲基化分析以及构建NKD2的真核表达载体，体外转染胃癌细胞后研究其对胃癌细胞体外以及小鼠异种移植瘤的生长、凋亡、细胞周期以及迁移侵袭能力的影响。此外，有文献报道NKD2基因参与Wnt信号通路的负性调控，我们将进一步分析NKD2基因与Wnt信号通路中重要功能蛋白质的相互作用。该课题按照预定实验计划顺利实施并取得一系列进展，通过研究证实NKD2基因在胃癌细胞以及人胃癌组织表达明显降低，该基因的表达降低并非为基因突变所致，而是与该基因启动子区的高甲基化有关。将NKD2基因的真核表达载体转染到胃癌细胞株后，明显抑制了胃癌细胞的细胞周期进程，细胞阻滞于G2/M期。体外过表达NKD2明显抑制了胃癌细胞的增殖、迁移与侵袭能力，抑制了裸鼠体内异种移植瘤的生长，但对胃癌细胞的凋亡影响不明显。通过对胃癌细胞的提取蛋白的免疫共沉淀实验证实，NKD2蛋白是与Wnt信号通路关键蛋白Dishevelled具有相互作用，参与了Wnt信号通路的负性调控。该项目实施期间培养了博士研究生5名，锻炼了课题组内青年科研人员，资助项目组人员与研究生出席国内外学术会议多人次。并在国际、国内学术刊物发表受本基金资助学术论文15篇。项目实施期间，申报胃癌发病机制与转化应用相关的发明专利1项，国家版权局计算机软件著作权2项。获得省部级科技奖项4项，获得各种荣誉多项。