Micro-RNA Molecules as Regulators of Diabetic Wound Healing

描述（由申请人提供）：本项目的目的是使用基因组学方法来鉴定和证明特定的microRNA分子阻碍糖尿病个体的伤口愈合，从而表明它们作为新型治疗靶点的潜力。 本项目通过解决以下研究领域来响应RFA-NR-12-002：a）阐明与慢性伤口易感性、发展、进展相关的基因组标志物/机制，和修复~ B）开发和测试旨在预防慢性伤口和/或加速愈合过程的基于基因组的干预。 糖尿病足溃疡是糖尿病的主要并发症之一，每年有成千上万的糖尿病患者因此而截肢。因此，对于改善这些慢性伤口的愈合的特异性和有效的治疗剂存在关键的未满足的需求。miRNA构成通过转录后基因沉默调节基因表达的非编码基因组物质，并且可以用合成的小分子药物中和。我们实验室的初步研究鉴定了一组在慢性伤口中选择性过表达的miRNA，其抑制体内急性伤口愈合，并且可能代表潜在的治疗靶点。 该项目的目标是测试DFU特异性miRNA的诱导通过抑制协调伤口愈合过程的基因而导致愈合障碍的假设，并确定用序列特异性抗miRNA寡核苷酸（sequence-specific anti-miRNA oligonucleotides）靶向它们是否逆转愈合障碍。 目的1是通过定量DFU组织活检中的miRNA，并使用DFU诱导的miRNA重建小鼠急性实验伤口中的慢性DFU，来表征DFU患者中诱导的miRNA对体外和体内愈合的抑制。 目的2是通过使用合成的靶向DFU特异性miRNAs的合成药物来定义DFU特异性miRNAs的功能作用，并逆转由患者DFU活检产生的原代细胞培养物和小鼠体内伤口愈合模型中的不愈合表型。 目的3：通过基因组学方法同时定量DFU组织中mRNA和miRNA基因表达谱，鉴定DFU特异性miRNA，并确定其下游靶点。通过该方法鉴定的新miRNA将通过目的1和2的方法进行验证。 这些研究是新颖的和重要的应用建立的miRNA策略和生物信息学分析的问题，验证潜在的新的治疗靶点慢性DFU。该项目的成功完成将导致未来的研究，通过临床前和临床试验开发候选疗法。 公共卫生相关性：本项目应用microRNA（miRNA）基因组学的最新突破，在体外实际患者伤口细胞和体内小鼠伤口模型中测试通过其合成模拟物和/或靶向特定miRNA分子是否可以逆转不愈合特性。该项目的成功完成将导致 在鉴定一类新的治疗候选物，miRNA分子，这是负责慢性糖尿病足溃疡愈合抑制。

DESCRIPTION (provided by applicant): The objective of this project is to use a genomics approach to identify and demonstrate that specific microRNA molecules impede wound healing in diabetic individuals, thus indicating their potential as novel therapeutic targets. This projet responds to RFA-NR-12-002 by addressing following areas of research interest: a) Elucidation of the genomic markers/mechanisms related to chronic wound susceptibility, development, progression, and repair~ b) Development and testing of genomic based interventions aimed at preventing chronic wounds and/or expediting the healing process. Diabetic foot ulcers (DFUs) are one of the major complications of diabetes mellitus leadin to lower extremity amputation for thousands of diabetic persons each year. Thus, there is a critical unmet need for specific and effective therapeutics to improve healing of these chronic wounds. miRNAs constitute non-coding genomic species that regulate gene expression through post-transcriptional gene silencing and can be neutralized with synthetic small-molecule drugs. Preliminary studies in our laboratory identified a set of miRNAs that are selectively over-expressed in chronic wounds, inhibit acute wound healing in vivo, and may represent potential therapeutic targets. The goals of this project are to test the hypothesis that induction of DFU-specific miRNAs causes healing impairment by repressing genes that coordinate wound healing process, and to determine if targeting them with antagomirs (sequence-specific anti-miRNA oligonucleotides) reverses the healing impairment. Aim 1 is to characterize inhibition of healing in vitro and in vivo by miRNAs identified as induced in DFU patients by quantifying miRNAs in tissue biopsies from DFUs, and using the DFU-induced miRNAs to reconstruct the chronic DFU in acute experimental wounds in mice. Aim 2 is to define the functional role of DFU-specific miRNAs by using synthetic antagomirs to target DFU-specific miRNAs and reverse the non-healing phenotype in primary cell cultures generated from patients' DFU biopsies and in the mouse wound healing model in vivo. Aim 3 is to identify DFU-specific miRNAs and determine their downstream targets specific for wound healing process by genomics approach of quantifying simultaneously mRNA and miRNA gene expression profiles of DFU biopsies. New miRNAs that are identified by this approach will be validated by the methods of Aims 1 and 2. These studies are novel and significant in applying established miRNA strategies and bioinformatics analysis to the problem of validating potential new therapeutic targets for chronic DFUs. Successful completion of this project will lead to future studies to develop candidate therapeutics through preclinical and clinical testing. PUBLIC HEALTH RELEVANCE: This project applies recent breakthroughs in the genomics science of micro RNA (miRNA) to test if targeting specific miRNA molecules by their synthetic mimics and/or antagomirs in actual patients' wound cells in vitro and mouse wound model in vivo may reverse non-healing characteristics. Successful completion of this project will result in identification of a new class of therapeutic candidates, miRNA molecules, that are responsible for the healing inhibition in chronic diabetic foot ulcers .