Inherited colorectal cancer risk variants: from association to biology

 描述（由申请方提供）：拟定研究的目的是辨别通过全基因组关联研究（GWAS）鉴定的结直肠癌（CRC）风险变体的功能和生物学相关性。在第一个融资期间，我们建立了一个功能表征管道，以调查CRC风险的机制基础。使用该管道，我们通过eQTL分析鉴定了8个GWAS区域的功能调控元件/增强子/启动子和8个GWAS区域的靶基因。为了跟上新的GWAS风险变体的发现速度，并进一步询问GWAS风险变体的机制和生物学相关性，我们现在提出以下具体目标。目标1：我们将建立在我们已经开发的成功的分子表征管道的基础上，并通过结合来自OncoArray研究的精细作图数据、来自10名健康受试者的正常结肠隐窝的全基因组染色质免疫沉淀和测序（ChIPseq）数据，并使用来自>1100个正常结肠上皮活检的RNA-seq数据应用全基因组eQTL分析。目标二：使用目标1的数据，我们将使用慢病毒系统在正常人3D结肠上皮类器官培养物中敲低或过表达候选风险靶基因，并检查对形态学，增殖，凋亡和常见信号传导途径的影响，然后通过免疫组织化学/荧光方法在正常组织中进行验证。我们将在CRC细胞系中通过CRISPR-Cas9方法敲除调控元件，然后进行RT-qPCR验证后，确认活性调控元件与靶基因之间的相关性。在没有鉴定出活性调控区的靶基因的情况下，我们将在CRC细胞系中通过CRISPR-Cas9方法敲除调控元件后鉴定候选靶基因，然后进行RNA-Seq eQTL分析。最后，在目标3中：我们将检验CRC风险变体导致结肠隐窝中过早衰老表型的假设。我们将确定风险变异负担和结肠隐窝中累积的DNA突变之间的相关性。将通过测量组蛋白H2 AX磷酸化、全基因组测序和通过定量PCR测量的端粒长度来评估DNA损伤。这项研究将提供深入了解遗传风险变异对结肠隐窝和CRC病因学的正常生物学的作用。

 DESCRIPTION (provided by applicant): The goal of the proposed study is to discern the functional and biological relevance of colorectal cancer (CRC) risk variants identified through genome wide association studies (GWAS). During the first funding period we established a functional characterization pipeline to investigate the mechanistic basis underlying CRC risk. Using this pipeline we identified functional regulatory elements/enhancers/promoters for 8 GWAS regions and target genes for 8 GWAS regions by eQTL analysis. To keep pace with the rate of discovery of novel GWAS risk variants and to further interrogate the mechanistic and biological relevance of GWAS risk variants we now propose the following Specific Aims. Aim 1: We will build upon the successful molecular characterization pipeline we have developed and identify additional novel functional regulatory regions/enhancers/promoters and target genes from GWAS risk regions through incorporation of fine mapping data from the OncoArray study, genome wide chromatin immunoprecipitation and sequencing (ChIPseq) data from normal colon crypts from 10 healthy subjects, and apply genome wide eQTL analyses using RNA-seq data from >1100 normal colon epithelial biopsies. Aim 2: Using data from Aim 1 we will knock down or over-express candidate risk target genes in normal human 3D colon epithelial organoid cultures using lentiviral systems and examine the effect on morphology, proliferation, apoptosis and common signaling pathways followed by validation in normal tissues by immunohistochemical/fluorescence approaches. We will confirm the correlation between active regulatory elements and target genes following knock out of regulatory elements by CRISPR-Cas9 methods in CRC cell lines followed by RT-qPCR validation. Where no target genes of active regulatory regions have been identified we will identify candidate target genes following knock out of regulatory elements by CRISPR-Cas9 methods in CRC cell lines followed by RNA-Seq eQTL analysis. Finally, in Aim 3: We will test the hypothesis that CRC risk variants lead to a premature aging phenotype in colon crypts. We will determine the correlation between risk variant burden and accumulated DNA mutations in colon crypts. DNA damage will be assessed by measuring histone H2AX phosphorylation, whole genome sequencing and telomere length measured by quantitative PCR. This study will provide insight into the role of genetic risk variants on normal biology of the colon crypt and CRC etiology.