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：
9304914
负责人：
GRAHAM CASEY
金额：
$ 63.48万
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/9304914
关键词：
11q23 19q13 1q41 20p12 8q24 APC gene 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 Chromosomes Colon Colorectal Cancer Data Development Embryo Enhancers Epithelial Etiology Event Fluorescent in Situ Hybridization Future Gene Expression Gene Targeting Generations Genes Genetic Enhancer Element 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 Regulator Genes Regulatory Element Research Risk Role SW480 Series Signal Pathway Spleen T-Lymphocyte Technology Transcript Transforming Growth Factor beta Transgenic Mice Translating Variant Wild Type Mouse Work base c-myc Genes cancer risk carcinogenicity chromosome conformation capture colon cancer cell line crypt cell embryonic stem cell experimental study 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 risk variant success tool transcription factor transcriptome transcriptome sequencing transmission process tumor

项目摘要

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）：只有当我们完全了解遗传风险关联的生物学后果时，才能实现全基因组关联研究（GWAS）的全部潜力。拟议研究的目标是确定经过验证的结直肠癌的基因靶点。 CRC) GWAS 风险增强剂使用一系列补充方法，并使用一种新颖的基于小鼠的体内方法开始确定风险增强剂在正常隐窝发育和 CRC 病因学中的生物学作用。在目标 1 中，我们识别染色体 1q41、3p14.1、8q24.21、11q23.1、15q13.3（3 个风险增强子）、18q21.1、19q13.11、19q21 和 20p12.3 上的 GWAS 位点内的 CRC 风险增强子。将通过基因组研究来识别这些 CRC 风险增强剂的新靶基因使用来自超过 1000 个正常结肠上皮活检的 RNA-Seq 数据进行广泛的 eQTL 分析，并通过 CRISPR/Cas9 介导的敲除 CRC 细胞系中的风险增强子，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。 -使用染色体构象捕获方法的靶基因相互作用我们将使用环化染色体构象捕获（4C）方法来识别和验证风险增强剂和靶基因之间的物理相互作用。 HCT116 和 SW480 CRC 细胞系将使用染色质构象捕获 (3C) 和荧光原位杂交 (FISH) 进一步验证特定增强子与靶基因的相互作用。在目标 3 中，我们将使用新型小鼠测试 CRC 风险增强子的生物学效应。将开发具有与已知局部目标的 3 个风险增强子 GWAS 区域（包括 15q13.3 上的多重增强子区域）相对应的选定人类 BAC 的模型系统。基因（8q24.21/cMYC/CCAT2、11q23.1/C11orf53/C11orf92/C11orf93 和 15q13.3/GREM1/FMN1/ax747968）。细胞将与野生型四倍体胚胎结合生成嵌合小鼠，其中整个胚胎本身源自修饰的 ES 细胞。使用转录组分析（RNA-Seq）分析风险和非风险 SNP 对目标基因转录水平的影响。将在这些小鼠的肠隐窝和非结肠细胞（例如肝脏、脾脏）中进行组织学研究，以检查风险增强子 SNP 对正常隐窝和非结肠细胞的影响。这些实验将在 Apc 野生型转基因小鼠以及携带 Apc 基因杂合无效突变的小鼠中进行。拟议的研究将深入了解 Apc 的生物学作用。肠隐窝和结直肠癌病因学中的风险增强剂风险增强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。