Inherited colorectal cancer risk variants: from association to biology

 描述(由申请人提供)：拟议研究的目标是识别通过全基因组关联研究(GWAS)确定的结直肠癌(CRC)风险变异的功能和生物学相关性。在第一个资助期内，我们建立了一个功能表征管道，以调查儿童权利公约风险的机制基础。利用该文库，我们通过eQTL分析确定了8个GWA区的功能调控元件/增强子/启动子以及8个GWA区的靶基因。为了跟上新的GWA型风险变异体的发现速度，并进一步探讨GWA型风险变异体的机制和生物学相关性，我们现在提出以下具体目标。目的1：我们将在我们开发的成功的分子表征流水线的基础上，通过整合来自OncoArray研究的精细定位数据、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&gt；1100正常结肠上皮活检的RNA-seq数据，从GWAs风险区域识别和鉴定其他新的功能调节区/增强子/启动子和靶基因。目的2：利用来自Aim 1的数据，我们将在使用慢病毒系统的正常人3D结肠上皮器官培养中敲除或过度表达候选风险靶基因，并检测其对形态、增殖、细胞凋亡和常见信号通路的影响，然后在正常组织中用免疫组织化学/荧光方法进行验证。我们将通过CRISPR-Cas9方法在结直肠癌细胞系中敲除调控元件后，确认活性调控元件与靶基因之间的相关性，然后进行RT-qPCR验证。在没有发现活性调控区域的靶基因的情况下，我们将通过CRISPR-Cas9方法敲除大肠癌细胞株中的调控元件，然后进行RNA-Seq eQTL分析，从而确定候选的靶基因。最后，在目标3：我们将测试结直肠癌风险变异导致结肠隐窝过早衰老表型的假设。我们将确定风险变异负担与结肠隐窝中累积的DNA突变之间的相关性。DNA损伤将通过测量组蛋白H_2AX磷酸化、全基因组测序和定量PCR测量端粒长度来评估。这项研究将深入了解遗传风险变异在结肠隐窝正常生物学和结直肠癌病因中的作用。

 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.