Biology of Colorectal Cancer Risk Enhancers

结直肠癌风险增强剂的生物学

• 批准号: 9081353
• 负责人: GRAHAM CASEY
• 金额: $ 7.73万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9081353
• 关键词: 1q41; 3p14.1; Affinity; Alleles; B-Lymphocytes; BMP2 gene; Bacterial Artificial Chromosomes; Biological; Biological Assay; Biological Models; Biological Testing; Biology; Biopsy; CRISPR/Cas technology; Cancer Etiology; Cells; Chromatin; Chromosome Mapping; Chromosomes; Colon; Colorectal Cancer; Data; Development; Embryo; Enhancers; Epithelial; Etiology; Event; Fluorescent in Situ Hybridization; Future; Gene Expression; Gene Targeting; Generations; Genes; Genetic Risk; Goals; Guidelines; HCT116 Cells; Human; In Vitro; Intervention; Intestinal Polyps; Knock-out; Linkage Disequilibrium; Liver; MADH7 gene; Maps; Mediating; Methods; Molecular Conformation; Mus; Play; Prevention; Regulatory Element; Research; Risk; Role; SW480; Series; Signal Pathway; Spleen; T-Lymphocyte; Technology; Transcript; Transforming Growth Factor beta; Transgenic Mice; Translating; Variant; Work; base; cancer genome; cancer risk; chromosome conformation capture; colon cancer cell line; crypt cell; embryonic stem cell; gene interaction; genome wide association study; genome-wide; histological studies; improved; in vivo; insight; intestinal crypt; mouse model; novel; null mutation; promoter; public health relevance; research study; risk variant; success; tool; transcription factor; transcriptome; transcriptome sequencing; transmission process; tumor

 DESCRIPTION (provided by applicant): The full potential of genome wide association studies (GWAS) will only be realized once we fully understand the biological consequences of genetic risk associations. The goal of the proposed study is to identify gene targets of validated colorectal cancer (CRC) GWAS risk enhancers using a series of complementary approaches and to begin to establish the biological role of risk enhancers in normal crypt development and CRC etiology using a novel in vivo murine-based method. This study builds upon our previous successes in identifying CRC risk enhancers within GWAS loci on chromosomes 1q41, 3p14.1, 8q24.21, 11q23.1, 15q13.3 (3 risk enhancers), 18q21.1, 19q13.11, 19q21 and 20p12.3. In Aim 1 we will identify novel target genes of these CRC risk enhancers by conducting genome wide eQTL analyses using RNA-Seq data from >1000 normal colon epithelial biopsies and by CRISPR/Cas9-mediated knock out of the risk enhancers in CRC cell lines followed by RNA-Seq eQTL analysis. In Aim 2 we will identify and validate risk enhancer-target gene(s) interactions using chromosome conformation capture methods. We will identify and validate the physical interaction between risk enhancers and target genes using the circularized chromosome conformation capture (4C) method using HCT116 and SW480 CRC cell lines. Specific enhancer-target gene interactions will be further validated using chromatin conformation capture (3C) and fluorescence in situ hybridization (FISH). In Aim 3 we will test the biological effect of CRC risk enhancers using a novel mouse model system. Mice will be developed that harbor selected human BACs corresponding to 3 risk enhancer GWAS regions (including the multiple enhancer region on 15q13.3) with known local target genes (8q24.21/cMYC/ CCAT2, 11q23.1/C11orf53/ C11orf92/ C11orf93 and 15q13.3/GREM1/ FMN1/ ax747968). BACs will be inserted into mouse ES cells and CRISPR/Cas9 technology will be used to introduce either risk or non-risk variants within risk enhancers. The modified ES cells will be combined with wild type tetraploid embryos to generate chimeric mice in which the entire embryo-proper was derived from the modified ES cells. The effects of the risk and non-risk SNPs on target gene transcript levels using transcriptome profiling (RNA-Seq) will be determined in these mice in intestinal crypts and non-colon cells (e.g. liver, spleen). Histological studies will be conducted to examine the effects of risk enhancer SNPs on normal crypt and intestine polyp/tumor development. These experiments will be carried out in transgenic mice that are wild-type for Apc, as well as mice that carry a heterozygous-null mutation in the Apc gene. The proposed research will provide insight into the biological role of risk enhancers in the intestinal crypt and CRC etiology and the discovery of risk enhancer target genes will provide tools for future early surveillance and prevention studies of CRC.

 描述（由申请人提供）：全基因组关联研究（GWAS）的全部潜力只有在我们完全理解遗传风险关联的生物学后果后才能实现。本研究的目的是使用一系列互补方法鉴定经验证的结直肠癌（CRC）GWAS风险增强子的基因靶点，并使用一种新的基于小鼠的体内方法开始建立风险增强子在正常隐窝发育和CRC病因学中的生物学作用。这项研究建立在我们以前成功鉴定染色体1 q41，3p14.1，8q24.21，11q23.1，15q13.3（3个风险增强子），18q21.1，19q13.11，19 q21和20p12.3上GWAS基因座内CRC风险增强子的基础上。在目标1中，我们将通过使用来自>1000个正常结肠上皮活检的RNA-Seq数据进行全基因组eQTL分析，并通过CRISPR/Cas9介导的CRC细胞系中风险增强子的敲除，随后进行RNA-Seq eQTL分析，来鉴定这些CRC风险增强子的新靶基因。在目标2中，我们将使用染色体构象捕获方法鉴定和验证风险增强子-靶基因相互作用。我们将使用HCT 116和SW 480 CRC细胞系，使用环化染色体构象捕获（4C）方法鉴定和验证风险增强子与靶基因之间的物理相互作用。将使用染色质构象捕获（3C）和荧光原位杂交（FISH）进一步验证特异性增强子-靶基因相互作用。在目标3中，我们将使用新的小鼠模型系统测试CRC风险增强剂的生物学效应。将开发具有选定的人BAC的小鼠，所述人BAC对应于具有已知局部靶基因（8q24.21/cMYC/CCAT 2、11q23.1/C11 orf 53/C11 orf 92/C11 orf 93和15q13.3/GREM 1/FMN 1/ ax747968）的3个风险增强子GWAS区域（包括15q13.3上的多增强子区域）。BAC将被插入小鼠ES细胞，CRISPR/Cas9技术将被用于在风险增强子中引入风险或非风险变体。修饰的ES细胞将与野生型四倍体胚胎组合以产生嵌合小鼠，其中整个胚胎-适当的来自修饰的ES细胞。将在这些小鼠的肠隐窝和非结肠细胞（例如肝、脾）中使用转录组分析（RNA-Seq）确定风险和非风险SNP对靶基因转录水平的影响。将进行组织学研究以检查风险增强子SNP对正常隐窝和肠息肉/肿瘤发展的影响。这些实验将在Apc野生型转基因小鼠以及Apc基因中携带异源无效突变的小鼠中进行。拟议的研究将提供深入了解风险增强剂在肠隐窝和CRC病因学中的生物学作用 而风险增强子靶基因的发现将为未来CRC的早期监测和预防研究提供工具。