MicroRNA-mediated regulation of ATG16L1 expression and autophagy

说明(申请人提供)ATG16L1最近在全基因组关联研究中被确定为克罗恩病的易感基因，从而在克罗恩病的发病机制中牵涉到自噬。虽然最强的遗传关联导致密码子300(T300A)的苏氨酸到丙氨酸的转变可能改变所产生的蛋白质的功能，而且初步研究尚未发现与克罗恩病相关的ATG16L1表达的变化，但使用表达低水平ATG16L1的低形态小鼠的研究表明，ATG16L1的表达减少可能显著改变其在肠道上皮细胞中的功能。总体而言，对ATG16L1和其他自噬基因的表达是如何调控的缺乏了解。MicroRNAs是一种21-24个核苷酸的非编码RNA分子，通过与转录产物3‘端非翻译区的互补序列结合来负向调节基因的表达，并指导mRNA的降解或蛋白质的翻译抑制。我们以前已经发现在克罗恩病和溃疡性结肠炎组织中差异表达的microRNAs。电子计算机分析发现，ATG16L13 3‘非编码区包含超过25个推测的microRNA结合位点和3个自然产生的SNPs，这些SNP映射到3个不同的推测的microRNA结合位点。我们的初步结果表明，ATG16L1 3‘非编码区在调节ATG16L1的表达中起着重要的作用。我们的长期目标是确定microRNAs在炎症性肠病(如克罗恩病)发病机制中的作用。本研究的目的是确定3‘非编码区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的表达方面发挥着重要作用，并且microRNA介导的ATG16L1表达的变化显著影响肠上皮细胞的自噬。我们将以以下两个具体目标来解决这一中心假说：(1)确定调控ATG16L1表达和随后的自噬和异噬的microRNA结合位点；(2)确定microRNA结合位点SNP对ATG16L1表达和随后的自噬和异噬的影响。这些目标将通过已建立的microRNA和自噬功能分析来实现，并使用体细胞基因打靶来产生携带microRNA结合位点SNPs的肠上皮细胞。这些结果有望产生重要的积极影响，因为microRNA对ATG16L1表达和自噬的调节提供了一种生理机制，通过该机制可以在细胞水平上调节自噬并影响疾病过程。这些结果将证实利用体细胞突变来研究SNP对3‘UTRmicroRNA结合部位功能的影响，并可能提供未来与microRNA相关的途径，从而可能成为自噬相关疾病的靶点。

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.