Biology of colorectal cancer risk enhancers

结直肠癌风险增强剂的生物学

• 批准号: 9411989
• 负责人: GRAHAM CASEY
• 金额: $ 59.92万
• 依托单位: UNIVERSITY OF VIRGINIA
• 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 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9411989
• 关键词: Biology; colorectal; cancer; risk; enhancers

PROJECT SUMMARY/ABSTRACT 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.

项目总结/摘要 全基因组关联研究（GWAS）的全部潜力只有在我们完全了解 遗传风险关联的生物学后果。这项研究的目的是确定基因 使用一系列互补的靶点验证结直肠癌（CRC）GWAS风险增强剂， 方法，并开始建立风险增强剂在正常隐窝发育中的生物学作用， 使用一种新的基于鼠的体内方法的CRC病因学。这项研究建立在我们以前的成功， 在染色体1 q41、3p14.1、8q24.21、11q23.1、15q13.3上的GWAS基因座内鉴定CRC风险增强子 （3个风险增强子）、18q21.1、19q13.11、19 q21和20p12.3。在目标1中，我们将鉴定新的靶基因， 这些CRC风险增强子通过使用来自>1000个 通过CRISPR/Cas9介导的CRC细胞中风险增强子的敲除， 系，然后进行RNA-Seq eQTL分析。在目标2中，我们将识别和验证风险增强子-靶基因 使用染色体构象捕获方法的相互作用。我们将确认并验证 使用环状染色体构象捕获的风险增强子和靶基因之间的相互作用 (4C)方法使用HCT 116和SW 480 CRC细胞系。特异性增强子-靶基因相互作用将是 使用染色质构象捕获（3C）和荧光原位杂交（FISH）进一步验证。在 目的3我们将使用一种新的小鼠模型系统来测试CRC风险增强剂的生物学效应。小鼠将被 开发了具有对应于3个风险增强子GWAS区域的选择的人类BAC（包括 15q13.3上的多个增强子区域）与已知的局部靶基因（8q24.21/cMYC/CCAT 2， 11q23.1/C11 orf 53/C11 orf 92/C11 orf 93和15q13.3/GREM 1/FMN 1/ ax747968）。BAC将被插入到 小鼠ES细胞和CRISPR/Cas9技术将被用于引入风险或非风险变体， 风险增强剂。修饰的ES细胞将与野生型四倍体胚胎结合以产生嵌合体胚胎。 小鼠，其中整个胚胎-适当来源于修饰的ES细胞。风险的影响， 使用转录组分析（RNA-Seq）确定靶基因转录水平上的非风险SNP， 这些小鼠在肠隐窝和非结肠细胞（例如肝、脾）中。将进行组织学研究， 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 实验将在野生型Apc的转基因小鼠以及携带Apc基因的小鼠中进行。 Apc基因中的杂合无效突变。这项拟议中的研究将深入了解生物学作用， 风险增强子在肠隐窝和CRC病因学中的作用以及风险增强子靶基因的发现将 为今后CRC的早期监测和预防研究提供工具。