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 只有一旦我们完全理解，基因组广泛关联研究（GWAS）的全部潜力才能实现 遗传风险关联的生物学后果。拟议的研究的目的是识别基因 经过验证的结直肠癌（CRC）GWAS风险增强子的靶标使用一系列互补 方法并开始确定风险增强子在正常地下隐窝发展中的生物学作用和 CRC病因使用一种新型的基于体内鼠的方法。这项研究以我们以前的成功为基础 识别GWAS基因座中的CRC风险增强子1Q41、3P14.1、8Q24.21、11Q23.1、15Q13.3的CRC风险增强子。 （3风险增强剂），18Q21.1，19Q13.11，19Q21和20P12.3。在AIM 1中，我们将确定新颖的目标基因 这些CRC风险增强子通过使用> 1000的RNA-seq数据进行基因组范围的EQTL分析。 正常结肠上皮活检以及CRISPR/CAS9介导的CRC细胞中的风险增强子的敲除 线然后进行RNA-seq EQTL分析。在AIM 2中，我们将确定并验证风险增强子目标基因（S） 使用染色体构象捕获方法的相互作用。我们将识别并验证物理 使用循环染色体构象捕获风险增强子与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 AIM 3我们将使用新型的小鼠模型系统测试CRC风险增强子的生物学效应。老鼠会 开发了选择与3个风险增强子GWAS区域相对应的人类BAC（包括 具有已知局部靶基因（8Q24.21/ CMYC/ CCAT2， 11Q23.1/ c11orf53/ c11orf92/ c11orf93和15q13.3/ grem1/ fmn1/ ax747968）。 BAC将插入 小鼠ES细胞和CRISPR/CAS9技术将用于引入风险或非风险变体 风险增强器。修饰的ES细胞将与野生型四倍体胚胎结合起来，以生成嵌合 整个胚胎培训器的小鼠源自修饰的ES细胞。风险的影响和 使用转录组分析（RNA-SEQ）在目标基因转录水平上的非风险SNP将在 这些小鼠在肠道隐窝和非彩色细胞中（例如肝脏，脾脏）。组织学研究将进行 检查风险增强子SNP对正常隐窝和肠道息肉/肿瘤发育的影响。这些 实验将在APC的野生型的转基因小鼠中进行，以及携带的小鼠 APC基因中的杂合无效突变。拟议的研究将洞悉生物学作用 肠道隐窝和CRC病因中的风险增强子以及风险增强子目标基因的发现将 为CRC的未来早期监视和预防研究提供工具。