Inherited colorectal cancer risk variants: from association to biology

 描述（由申请人提供）：拟议研究的目标是辨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。在第一个资助期间，我们建立了功能表征管道来研究 CRC 风险的机制基础。使用该流程，我们通过 eQTL 分析确定了 8 个 GWAS 区域的功能调控元件/增强子/启动子以及 8 个 GWAS 区域的靶基因。为了跟上新型 GWAS 风险变异的发现速度，并进一步探讨 GWAS 风险变异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：我们将在已开发的成功分子表征流程的基础上，通过结合 OncoArray 研究的精细图谱数据、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，识别来自 GWAS 风险区域的其他新型功能调节区/增强子/启动子和靶基因，并使用来自 >1100 个正常结肠上皮的 RNA-seq 数据应用全基因组 eQTL 分析活检。目标 2：利用目标 1 的数据，我们将使用慢病毒系统敲低或过度表达正常人 3D 结肠上皮类器官培养物中的候选风险靶基因，并检查对形态、增殖、凋亡和常见信号通路的影响，然后通过免疫组织化学/荧光方法在正常组织中进行验证。我们将通过 CRC 细胞系中的 CRISPR-Cas9 方法敲除调控元件，然后进行 RT-qPCR 验证，确认活性调控元件与靶基因之间的相关性。如果尚未鉴定出活性调控区域的靶基因，我们将通过 CRC 细胞系中的 CRISPR-Cas9 方法敲除调控元件，然后进行 RNA-Seq eQTL 分析来鉴定候选靶基因。最后，在目标 3 中：我们将检验 CRC 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 H2AX 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对结肠隐窝正常生物学和结直肠癌病因学的作用。

 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.