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

• 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
• 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' 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

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.

由于伤口愈合不足，导致慢性伤口是一个重大的，而且由于 糖尿病和我们老龄化人口的不断增长。对组织损伤的先天免疫反应至关重要 伤口修复。单核细胞/巨噬细胞，均被招募和组织居民，至关重要的秘密因素 早期增生剂和再生伤口愈合阶段的介体。很少关注 转录后调控对伤口局部巨噬细胞中基因表达的影响。这是一个专业 由于大多数有影响力的巨噬细胞衍生 产品由带有Au富元素（ARES）和许多microRNA（miRNA）的mRNA编码 在其3'-非翻译区域（3'UTR）中结合位点。也就是说，转录激活触发通常是 特别是，RNA结合蛋白（RBP）保护脆弱 3'Utrs来自miRNA结合和随之而来的翻译停滞（和/或mRNA降解）。我们有 最近证明巨噬细胞β2整联蛋白参与导致RBP的动态调制 HUR保护了许多3'Utr是含有含量的mRNA，免受降解或翻译块。我们有 表明巨噬细胞HUR基因删除的小鼠在多个伤口模型中具有修复缺陷。在动态中 时尚，HUR具有缓解miRNA介导的基因表达约束的能力。我们的假设是 一组伤口愈合促进基因的表达，无论是重叠还是在招募的组织中与众不同 居民巨噬细胞是由miRNA介导的翻译块的Hur依赖性释放驱动的。我们有 生成独特的，特定于巨噬细胞的翻译分析工具，现在建议：（1）记录 候选者的存在，并定义了正在进行主动的，依赖性翻译的小巨噬细胞mRNA 在早期伤口愈合反应中，使用翻译核糖体亲和力纯化（TRAP）测定法 核糖体融合蛋白L10A-EGFP的下拉，以髓样细胞特异性方式表达， 类型或基因删除小鼠； （2）定义靶向那些陷阱定义的mRNA的miRNA，用biotin-3'utr FACS选择的L10A-EGFP+的核糖螺旋桨螺旋桨，伤口局部巨噬细胞提取物，证实了它们的 双重荧光素酶3'UTR记者测定中的翻译抑制作用； （3）释放miRNA介导的 对伤口愈合促进的mRNA的翻译约束，局部应用miRNA目标位点阻滞剂 寡核苷酸干扰与特定mRNA 3'UTR靶位点结合的寡核苷酸，在背面全厚度 省和早期打孔器伤口模型，评估伤口区域，重新上皮化，血运重建和 成纤维细胞重生。这些分子和临床前方法针对基因表达调节 伤口响应性巨噬细胞的切换将提供机械洞察力，并具有治疗意义。