MicroRNA-mediated regulation of ATG16L1 expression and autophagy

MicroRNA 介导的 ATG16L1 表达和自噬调节

• 批准号: 8309034
• 负责人: JOHN KWON
• 金额: $ 7.8万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8309034
• 关键词: 3' Untranslated Regions; Address; Alanine; Autophagocytosis; Binding; Binding Sites; Biological Assay; Cell physiology; Codon Nucleotides; Computer Simulation; Crohn' s disease; Disease; Disease susceptibility; Epithelial Cells; Fostering; Future; Gene Expression; Gene Targeting; Genes; Goals; Inflammatory Bowel Diseases; Intestines; Knowledge; Mediating; Messenger RNA; MicroRNAs; Mission; Mus; Mutation; Nucleotides; Outcome; Pathogenesis; Physiological; Play; Process; Proteins; Public Health; Regulation; Reporting; Research; Role; Single Nucleotide Polymorphism; Single Nucleotide Polymorphism Map; Site; Somatic Cell; Susceptibility Gene; Threonine; Tissues; Transcript; Translational Repression; Ulcerative Colitis; Untranslated RNA; base; genetic association; genome wide association study; innovation; mRNA Transcript Degradation; therapeutic target

DESCRIPTION (provided by applicant) ATG16L1 was recently identified in genome-wide association studies as a Crohn's disease susceptibility gene, thereby implicating autophagy in the pathogenesis of Crohn's disease. While the strongest genetic association results in a threonine to alanine transition at codon 300 (T300A) that may alter function of the resultant protein and initial studies have not yet identified Crohn's disease-associated changes in ATG16L1 expression, studies using hypomorphic mice expressing low ATG16L1 levels indicate that the reduced expression of ATG16L1 may significantly alter its function in intestinal epithelial cells. Overall, there is a lack of knowledge of how the expression of ATG16L1 and other autophagy genes are regulated. MicroRNAs are 21-24 nucleotide noncoding RNA molecules that negatively regulate gene expression by binding to complementary sequences in the 3'untranslated region (3'UTR) of mRNA transcripts and directing either mRNA degradation or protein translational inhibition. We have previously identified microRNAs that are differentially-expressed in Crohn's disease and ulcerative colitis tissues. An in silico analysis found that the ATG16L1 3'UTR contains over 25 putative microRNA binding sites and 3 naturally occurring SNPs that map to 3 distinct putative microRNA binding sites. Our preliminary results indicate that the ATG16L1 3'UTR plays a significant role in regulating ATG16L1 expression. Our long-term goal is to determine the role of microRNAs in the pathogenesis of inflammatory bowel diseases, such as Crohn's disease. The objective here is to determine the influence of 3'UTR microRNA binding sites in the regulation of ATG16L1 expression and function. Our central hypothesis is that microRNA play a significant role in regulating ATG16L1 expression and that microRNA-mediated alterations in ATG16L1 expression significantly impact autophagy in intestinal epithelial cells. We will address this central hypothesis with the following two specific aims: (1) To identify microRNA binding sites regulating ATG16L1 expression and subsequent autophagy and xenophagy; (2) To determine microRNA binding site SNP effects on ATG16L1 expression and subsequent autophagy and xenophagy. These aims will be pursued using established microRNA and autophagy functional assays and generating intestinal epithelial cells harboring the microRNA binding site SNPs using somatic cell gene targeting. Such results are expected to have an important positive impact because the determination of microRNA regulation of ATG16L1 expression and autophagy provides a physiologic mechanism by which autophagy can be regulated at a cellular level and influence disease processes. These results will confirm the utility of somatic cell mutations to study SNP influences on 3'UTR microRNA binding site function and may provide future microRNA-associated avenues by which autophagy-associated diseases may be targeted.

最近在全基因组关联研究中鉴定出ATG16L1的描述（由申请人提供）是克罗恩病敏感性基因，从而暗示了自噬在克罗恩病的发病机理中。虽然最强的遗传关联导致在密码子300（T300A）处导致苏氨酸与可能改变所得蛋白质功能和初步研究的功能的丙氨酸过渡，但尚未确定Crohn疾病相关的ATG16L1表达的变化，但使用表达低ATG16L1水平的近形小鼠表达了ATG16 LINT的表达良好的表达，这表明ATG16L的表达能力降低了。总体而言，缺乏了解如何调节ATG16L1和其他自噬基因的表达。 microRNA是21-24个核苷酸非编码RNA分子，通过与mRNA转录物3'untranslated区域（3'UTR）的互补序列结合并指导mRNA降解或蛋白质翻译抑制，对基因表达进行负调节基因表达。我们先前已经鉴定出在克罗恩病和溃疡性结肠炎组织中差异表达的microRNA。一硅分析发现，ATG16L1 3'UTR包含超过25个推定的microRNA结合位点和3个天然存在的SNP，这些SNP映射到3个不同的假定microRNA结合位点。我们的初步结果表明，ATG16L1 3'UTR在调节ATG16L1表达中起重要作用。我们的长期目标是确定microRNA在炎症性肠病的发病机理中的作用，例如克罗恩病。这里的目的是确定3'UTR microRNA结合位点在ATG16L1表达和功能调节中的影响。我们的中心假设是，microRNA在调节ATG16L1表达中起重要作用，并且MicroRNA介导的ATG16L1表达的改变显着影响肠上皮细胞的自噬。我们将以以下两个特定目的来解决这一中心假设：（1）确定调节ATG16L1表达的microRNA结合位点以及随后的自噬和Xenophagy； （2）确定microRNA结合位点SNP对ATG16L1表达以及随后的自噬和异种量的影响。这些目标将使用已建立的microRNA和自噬功能测定法，并生成带有MicroRNA结合位点SNP的肠上皮细胞，该细胞使用体细胞基因靶向靶向。预计此类结果将产生重要的积极影响，因为测定ATG16L1表达和自噬的microRNA调节提供了一种生理机制，可以在细胞水平上调节自噬并影响疾病过程。这些结果将证实体细胞突变研究SNP对3'UTR microRNA结合位点功能的影响，并可能提供未来与MicroRNA相关的途径，通过这些途径，可以针对自噬相关疾病。