CDH1基因遗传变异与大肠癌的相关性及其介导上皮-间质转化进程的功能机制研究

Colorectal cancer(CRC) is one of the most common malignant tumors.Genome-wide association analysis located one of the susceptibility genes of CRC in 16q22 (CDH1 gene region).By bioinformatics analysis,we found that SNP rs16260 located in the promoter region of the CDH1 lies just at the binding site of ZEB1 and CDH1.Previous study has confirmed that the ZEB1 binds to the CDH1 promoter and down-regulates the expression of CDH1. Therefore,it is assumed that the existence of rs16260 may affect the bingding effiency of ZEB1 and CDH1 promoter region,thereby reducing CDH1 expression and promoting EMT procedure in intestinal epithelium and tumorigenesis.Through some analysis means such as fine-mapping haplotype analysis,human genome re-sequencing technology,electrophoretic mobility shift assay(EMSA),chromatin immunoprecipitation assay(CHIP) and chromosome conformation capture analysis(3C),we can pinpoint the function domain of CDH1 gene,analyze its role in EMT procedure of CRC,explore the relationship between the function domain and tumor grade,infiltration,lymphatic metastasis,chemotherapeutic sensitivity.The study will establish analysis process of functional tumor-associated SNPs and discover new bio-markers for colorectal cancer.

全基因组关联分析(GWAS)研究已识别出部分大肠癌易感基因，在后-GWAS时代对易感基因的功能验证成为功能基因组学的研究热点。16q22，即CDH1基因的所在区域，是GWAS发现的大肠癌易感基因之一。而对于16q22功能位点的精确定位及其功能验证尚未见报道。前期研究显示ZEB1与CDH1启动子区域结合可以下调CDH1的表达水平，进一步促进大肠癌上皮-间质转化进程。我们发现位于CDH1启动子区域的基因多态性 rs16260(A/C) SNP 正好处于ZEB1与CDH1的结合区域，并且荧光素酶报告基因实验显示该位点确实存在生物学功能。对该SNP的体内，体外功能验证及其与疾病预后的关系是本课题研究的主要内容。本研究将通过单倍域精确定位分析,人群基因组重测序技术精确定位CDH1基因上的功能位点，并将构建CDH1启动子缺失小鼠模型，以明确该SNP在大肠癌发生及其上皮-间质转化过程中所起的作用。

国内外的全基因组关联分析（GWAS）已经发现了数十个大肠癌易感位点，对易感位点的具体致病机制研究是后GWAS时代的研究热点。本课题组首次发现，在大肠上皮组织中，位于人类染色质16q22.1区域CDH1基因附近大肠癌易感位点的不同基因型与邻近基因ZFP90的表达水平密切相关，进而通过染色质构象捕获证实了ZFP90启动子区与CDH1基因内含子区域存在染色质远距离调控。从人大肠癌标本、体外细胞实验和体内动物实验等方面的研究发现，ZFP90在人结肠癌组织中表达上升，并且与大肠癌的肿瘤分期分级以及患者预后相关。ZFP90通过转录抑制调控了下游基因BMP4和IHH，进而增强了大肠上皮细胞的干性。在ZFP90敲除动物模型中，通过建立AOM诱导的大肠癌模型，ZFP90敲除组小鼠的肿瘤大小和数量显著小于对照野生型小鼠。本研究系统阐释了大肠癌易感位点16q22.1的促癌分子学机制，为大肠癌的诊治提供了新的证据。