Biology of colorectal cancer risk enhancers

结直肠癌风险增强剂的生物学

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要只有当我们完全理解后，才能充分发挥全基因组关联研究 (GWAS) 的潜力遗传风险关联的生物学后果。拟议研究的目标是鉴定基因使用一系列互补的方法，验证结直肠癌 (CRC) GWAS 风险增强剂的目标方法并开始确定风险增强剂在正常隐窝发育中的生物学作用和使用一种新型的基于小鼠的体内方法进行 CRC 病因学研究。这项研究建立在我们之前的成功基础上识别染色体 1q41、3p14.1、8q24.21、11q23.1、15q13.3 上 GWAS 位点内的 CRC 风险增强子（3 个风险增强剂）、18q21.1、19q13.11、19q21 和 20p12.3。在目标 1 中，我们将鉴定新的靶基因通过使用超过 1000 个样本的 RNA-Seq 数据进行全基因组 eQTL 分析，这些 CRC 风险增强剂正常结肠上皮活检并通过 CRISPR/Cas9 介导敲除 CRC 细胞中的风险增强子随后进行 RNA-Seq eQTL 分析。在目标 2 中，我们将识别并验证风险增强剂目标基因使用染色体构象捕获方法进行相互作用。我们将识别并验证物理使用环状染色体构象捕获风险增强剂和靶基因之间的相互作用 (4C)使用HCT116和SW480 CRC细胞系的方法。特定的增强子-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。在目标 3 我们将使用新型小鼠模型系统测试 CRC 风险增强剂的生物效应。老鼠将会开发出包含与 3 个风险增强 GWAS 区域相对应的选定人类 BAC（包括 15q13.3 上的多个增强子区域），具有已知的局部靶基因（8q24.21/cMYC/CCAT2， 11q23.1/C11orf53/C11orf92/C11orf93 和 15q13.3/GREM1/FMN1/ax747968）。 BAC 将被插入小鼠 ES 细胞和 CRISPR/Cas9 技术将用于在体内引入风险或非风险变异风险增强剂。修饰后的ES细胞将与野生型四倍体胚胎结合产生嵌合体小鼠的整个胚胎本身都来自改良的 ES 细胞。风险的影响和使用转录组分析 (RNA-Seq) 确定目标基因转录本水平上的非风险 SNP 这些小鼠的肠隐窝和非结肠细胞（例如肝、脾）。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/肿瘤发育的影响。这些实验将在 Apc 野生型转基因小鼠以及携带 Apc 的小鼠中进行 Apc 基因杂合无效突变。拟议的研究将深入了解其生物学作用肠隐窝和结直肠癌病因学中风险增强剂的研究以及风险增强剂靶基因的发现将为未来结直肠癌的早期监测和预防研究提供工具。