Micro-RNA Molecules as Regulators of Diabetic Wound Healing

描述(申请人提供)：这个项目的目标是使用基因组学方法来识别和证明特定的microRNA分子阻碍糖尿病患者的伤口愈合，从而表明它们作为新的治疗靶点的潜力。本项目响应RFA-NR-12-002，涉及以下研究领域：a)阐明与慢性伤口易感性、发展、进展和修复相关的基因组标记/机制；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措施。糖尿病足溃疡(DFU)是糖尿病的主要并发症之一，每年导致数以千计的糖尿病患者截肢。因此，需要一种特殊有效的治疗方法来改善这些慢性伤口的愈合，这一需求尚未得到满足。MiRNAs构成非编码基因组物种，通过转录后基因沉默来调节基因表达，并可被合成的小分子药物中和。我们实验室的初步研究发现了一组在慢性伤口中选择性过度表达的miRNAs，它们在体内抑制急性伤口愈合，可能是潜在的治疗靶点。该项目的目标是验证这样的假设，即诱导DFU特异性的miRNAs通过抑制协调伤口愈合过程的基因而导致愈合障碍，并确定以Anagomir(序列特异性的反miRNA寡核苷酸)靶向它们是否逆转了愈合障碍。目的1通过定量DFU组织活检组织中的miRNAs，并利用DFU诱导的miRNAs重建小鼠急性实验性创面的慢性DFU模型，研究DFU患者体内诱导的miRNAs对创面愈合的抑制作用。目的2是通过使用合成的反义体靶向DFU特异的miRNAs来确定DFU特异性miRNAs的功能作用，并逆转患者DFU活检产生的原代细胞培养中的不可愈合表型和体内小鼠伤口愈合模型中的不可愈合表型。目的3通过同时定量DFU活检组织的mRNA和miRNA基因表达谱的基因组学方法，鉴定DFU特异的miRNAs，并确定其在伤口愈合过程中的下游靶点。通过这种方法识别的新miRNAs将通过AIMS 1和AIMS 2的方法进行验证。这些研究在将已建立的miRNA策略和生物信息学分析应用于验证潜在的慢性DFU新治疗靶点的问题上具有新颖和重要的意义。该项目的成功完成将导致未来通过临床前和临床试验开发候选疗法的研究。

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.