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.

项目摘要/摘要 全基因组关联研究的全部潜力只有在我们完全了解了 遗传风险关联的生物学后果。这项拟议的研究的目标是识别基因 使用一系列补充性的GWA型结直肠癌(CRC)风险增强剂的靶点 并开始确定风险增强剂在正常隐窝发育和修复中的生物学作用。 使用一种基于小鼠体内的新方法进行结直肠癌病因学研究。这项研究建立在我们以前在 在染色体1q41、3p14.1、8q24.21、11q23.1、15q13.3的GwA基因座中发现结直肠癌风险增强子 (3个风险增强剂)、18q21.1、19q13.11、19q21和20p12.3。在目标1中，我们将识别新的靶基因 这些CRC风险增强剂通过使用来自&gt；1000的RNA-Seq数据进行全基因组eQTL分析来实现 正常结肠上皮活检及CRISPR/Cas9介导的结直肠癌细胞风险增强子的敲除 随后进行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(S) 使用染色体构象捕捉方法的相互作用。我们将识别并验证物理 利用环化染色体构象捕获技术研究风险增强剂与靶基因的相互作用 (4C)以HCT116和SW480结肠癌细胞株为研究对象。特定的增强子-靶基因相互作用将是 通过染色质构象捕捉(3C)和荧光原位杂交(FISH)进一步验证。在……里面 目的3利用一种新的小鼠模型系统，测试结直肠癌风险增强剂的生物学效应。老鼠会成为 开发了与3个风险增强子GWAS区域相对应的精选人类BAC(包括 位于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将被插入到 小鼠胚胎干细胞和CRISPR/Cas9技术将用于在 风险增强剂。将改良的ES细胞与野生型四倍体胚胎相结合产生嵌合体 小鼠的整个胚胎本身都是从改良的ES细胞中衍生出来的。风险和风险的影响 将使用转录组分析(RNA-Seq)确定目标基因转录水平上的非风险SNPs 这些小鼠在肠道隐窝和非结肠细胞(如肝、脾)中。将进行组织学研究，以 研究风险增强子SNPs对正常隐窝和肠息肉/肿瘤发生的影响。这些 实验将在野生型APC转基因小鼠以及携带APC基因的小鼠身上进行。 APC基因杂合性零突变。这项拟议的研究将提供对生物作用的洞察。 肠隐窝的风险增强剂和结直肠癌病因学以及风险增强剂靶基因的发现将 为今后的早期监测和预防研究提供工具。