通过表达数量性状座位分析确定结直肠癌新的易感基因及功能

Since the advent of high-density genotyping arrays, genome-wide association studies (GWAS) have been widely used to identify a number of loci associated with a multitude of disease traits. The vast majority of GWAS tag SNPs located in the intergenic or intronic regions. Identification of genes affected by causal variants and the mechanism by which genotype influences phenotype have been major challenges. In our previous study, we performed expression quantitative trait locus-based analyses (eQTLs) using information provided by the Cancer Genome Atlas and National Human Genome Research Institute in CRC tissues. Analysis of the eQTLs of CRC risk loci resulted in the discovery of one variant that was significantly associated with gene expression (CCDC12). It should be noted that identifying an eQTL provides only indirect evidence of a link between genotype and gene transcription. Previous studies have found that CCDC12 gene expression level in cancer tissue was significantly higher than normal tissue. Silence of CCDC12 gene can significantly inhibit the invasion and metastasis of tumor cells. In present study, we will study the function of CCDC12 gene in vitro and in vivo. Using the gene expression chip and protein chip, we also plan to find its possible target or pathway. This study will elucidate the role and mechanism of CCDC12 affecting the invasion and metastasis of CRC, which might provide the basis for possible new treatment options and ideas.

全基因组关联研究确定了多个结直肠癌易感SNP位点，但多数位点位于内含子区、基因间区。如何确定其调控的靶基因和靶基因的功能研究是后续研究的关键。表达数量性状座位(eQTL)分析是解决这一难题的有效手段。我们的前期研究中，对美国国立基因组研究所收录的结直肠易感 SNPs进行eQTL分析，发现全新的肿瘤相关基因CCDC12，其在结直肠癌发生发展中的作用及机制尚无研究。前期研究发现CCDC12基因在癌组织中表达水平显著高于对照癌旁组织；下调CCDC12基因，可以显著抑制肿瘤细胞增殖侵袭；CCDC12基因可能为新的肿瘤相关癌基因。本研究将进行体内体外实验研究CCDC12基因功能，同时利用基因表达芯片和蛋白质芯片寻找其可能的作用靶点或通路，深入探讨其肿瘤相关分子机制。最终为结直肠癌发生分子机制的阐明及寻找有效的治疗靶点提供新的线索。

通过整合表达定量特征位点(eQTL)分析发现，rs8180040与结肠腺癌(COAD)患者中的CCDC12显著相关。免疫组化染色和蛋白印迹证实CCDC12在COAD组织中高表达（IHC 51/75 vs 8/75，WB 10/12 vs 2/12）。这与TCGA数据库的RNA-Seq数据一致（P <0.001）。我们选择了SW480和LOVO细胞系敲低CCDC12，而HCT116和SW480-KD细胞系进行了上调，然后进行功能研究，并在BALB/c小鼠中建立异种移植肿瘤模型。敲除CCDC12可以显著降低结肠癌细胞的增殖、迁移、侵袭和致瘤性，而外源性过表达CCDC12则有相反的效果。我们借助4×plex Isobaric Tags for Relative and Absolute Quantitation(iTRAQ)检测来确定其功能和潜在的调控机制，iTRAQ检测结果表明，CCDC12的过表达会改变黏附连接通路上的蛋白质。随后在SW480细胞中过表达Snail并顺序敲减CCDC12，我们发现Snail的过表达并没有显著改变SW480细胞中CCDC12的水平，而敲减CCDC12则会降低Snail的水平。CCDC12在COAD的肿瘤发生、发展和侵袭中起着重要作用，可能通过调节Snail途径影响结肠癌细胞的上皮间充质转化过程。