Competitive macrophage microRNA-RNA binding protein interactions in wound repair

伤口修复中竞争性巨噬细胞 microRNA-RNA 结合蛋白相互作用

基本信息

批准号：
9439844
负责人：
JEFFREY R. BENDER
金额：
$ 32.24万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-20 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9439844
关键词：
3&apos Untranslated Regions Address Affinity Chromatography Appearance Area Attention Beds Binding Binding Sites Biological Assay Biotin Blood capillaries Cells Chimeric Proteins Chronic Complex Consequentialism Defect Diabetes Mellitus Dorsal Ear Economic Burden Elderly Elements Excision Fibroblasts Gelatinase B Gene Expression Generations Genes Health HuR protein Immune Incidence Individual Inflammation Inflammatory Inflammatory Response Influentials Injury Innate Immune Response Integrins Knockout Mice Luciferases Mediating Mediator of activation protein Messenger RNA MicroRNAs Modeling Molecular Morbidity - disease rate Multiple Trauma Mus Myeloid Cells Natural Immunity Natural regeneration Oligonucleotides Operative Surgical Procedures Pathway interactions Phase Phenotype Play Porifera Process Production Proteins RNA RNA-Binding Proteins Recruitment Activity Reporter Ribosomes Role Site Source Surgical wound Therapeutic Thick Tissues Transcript Transcriptional Activation Transcriptional Regulation Translating Translations Untranslated Regions Ursidae Family Vascular Endothelial Growth Factors Venous Insufficiency Width Work Wound Healing aging population capillary chronic wound diabetic economic cost gene repair healing implantation improved insight mRNA Transcript Degradation macrophage monocyte mortality novel pre-clinical protective effect regenerative repaired response tool wound

项目摘要

Inadequate wound healing, resulting in chronic wounds, is a major and increasing U.S. health problem, due to the rising incidence of diabetes and our aging population. Innate immune responses to tissue injury are critical to wound repair. Monocyte/macrophages, both recruited and tissue-resident, secrete factors that are critical mediators of the early proliferative and regenerative wound healing phases. Little attention has been paid to posttranscriptional regulatory influences on gene expression in wound localized macrophages. This is a major checkpoint in macrophage-dependent wound repair, since a majority of influential macrophage-derived products are encoded by mRNAs that bear both AU-rich elements (AREs) and numerous microRNA (miRNA) binding sites in their 3'-untranslated region (3'UTR). That is, a transcriptional activation trigger is often insufficient for adequate gene expression. In particular, RNA-binding proteins (RBPs) protect vulnerable 3'UTRs from miRNA binding and consequent translational arrest (and/or mRNA degradation). We have recently demonstrated that macrophage β2 integrin engagement results in dynamic modulation of the RBP HuR, which protects numerous 3'UTR ARE-bearing mRNAs from degradation or translation blocks. We have shown that macrophage HuR gene-deleted mice have repair defects in multiple wound models. In dynamic fashion, HuR has the ability to relieve miRNA-mediated gene expression constraints. Our hypothesis is that expression of a set of wound healing-promoting genes, both overlapping and distinct in recruited vs. tissue- resident macrophages, is driven by HuR-dependent release of miRNA-mediated translation blocks. We have generated unique, macrophage-specific translational profiling tools, and now propose to: (1) document the presence of candidate, and define novel, macrophage mRNAs undergoing active, HuR-dependent translation in early wound healing responses, using the Translating Ribosome Affinity Purification (TRAP) assay, with pulldowns of the ribosomal fusion protein L10a-EGFP, expressed in myeloid cell-specific fashion, in HuR wild- type or gene-deleted mice; (2) define miRNAs that target those TRAP-defined mRNAs, with biotin-3'UTR riboprobe pulldowns of FACS-sorted L10a-EGFP+, wound localized macrophage extracts, confirming their translation-repressing effects in dual luciferase 3'UTR reporter assays; and (3) release the miRNA-mediated translational constraint on wound healing-promoting mRNAs, topically applying miRNA target site blocker oligonucleotides which interfere with binding to specific mRNA 3'UTR target sites, in dorsal full thickness excisional and ear punch hole wound models, assessing wound area, re-epithelialization, revascularization and fibroblast repopulation. These molecular and preclinical approaches, directed at a gene expression regulatory switch in wound-responsive macrophages, will provide mechanistic insight with therapeutic implications.

伤口愈合不充分导致慢性伤口是美国一个主要且日益严重的健康问题，原因是糖尿病发病率的上升和人口老龄化对组织损伤的先天免疫反应至关重要。单核细胞/巨噬细胞（无论是募集的还是组织驻留的）都分泌对伤口修复至关重要的因子。早期增殖和再生伤口愈合阶段的介质很少受到关注。转录后调控对伤口局部巨噬细胞基因表达的影响。巨噬细胞依赖性伤口修复中的检查点，因为大多数有影响力的巨噬细胞衍生产品由含有富含 AU 的元件 (ARE) 和大量 microRNA (miRNA) 的 mRNA 编码其 3'-非翻译区 (3'UTR) 中的结合位点通常是转录激活触发器。尤其是，RNA 结合蛋白 (RBP) 不足以保护脆弱的基因。来自 miRNA 结合和随后的翻译停滞（和/或 mRNA 降解）的 3'UTR。最近证明巨噬细胞 β2 整合素参与导致 RBP 的动态调节 HuR，它可以保护许多带有 3'UTR 的 mRNA 免遭降解或翻译阻断。研究表明，巨噬细胞 HuR 基因缺失的小鼠在多个伤口模型中存在修复缺陷。时尚的是，HuR 有能力缓解 miRNA 介导的基因表达限制。一组促进伤口愈合的基因的表达，在募集和组织中既有重叠又有不同- 常驻巨噬细胞，是由 miRNA 介导的翻译块的 HuR 依赖性释放驱动的。独特生成的巨噬细胞特异性翻译分析工具，现在建议：（1）记录候选者的存在，并定义新的巨噬细胞 mRNA 正在经历活跃的 HuR 依赖性翻译在早期愈合伤口反应中，使用翻译核糖体亲和纯化 (TRAP) 测定，核糖体融合蛋白 L10a-EGFP 的下拉，以骨髓细胞特异性方式表达，在 HuR 野生中类型或基因删除的小鼠；(2) 定义针对那些 TRAP 定义的 mRNA 的 miRNA，带有生物素-3'UTR FACS 分选的 L10a-EGFP+、伤口局部巨噬细胞提取物的核糖探针下拉，证实了它们的双荧光素酶 3'UTR 报告基因检测中的翻译抑制作用；以及 (3) 释放 miRNA 介导的对伤口愈合促进 mRNA 的翻译限制，局部应用 miRNA 靶位点阻断剂干扰与特定 mRNA 3'UTR 靶位点结合的寡核苷酸，位于背侧全层切除和耳穿孔伤口模型，评估伤口面积、上皮再形成、血管重建和这些分子和临床前方法针对基因表达调控。伤口反应性巨噬细胞的转变将提供具有治疗意义的机制见解。