microRNA regulated processes in keratinocytes upon exposure to sulfur mustard: modulation by mimics und anti-miRs

接触硫芥后角质形成细胞中的 microRNA 调节过程：模拟物和抗 miR 的调节

• 批准号: 424562951
• 负责人: Professor Dr. Christian Ries
• 金额: --
• 依托单位: Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/424562951?language=en
• 关键词: microRNA; regulated; processes; keratinocytes; upon

Sulfur mustard (SM) is a highly toxic warfare agent that represents a continuous threat in various crisis regions, particularly through activities of terroristic groups. Exposure of the skin with SM evokes severe tissue damage and defects in wound healing leading to long-term hospitalization of patients. The underlying molecular and cellular pathomechanisms are poorly investigated. Up to now there is no pharmacological therapy available for the treatment of SM-induced defects in would healing. Own results from in vitro studies in skin cells provided evidence that SM affects the expression of microRNAs (miRNAs) and down-stream pathways and cell functions in keratinocytes. miRNAs are small ubiquitous RNA molecules that bind to specific mRNAs and inhibit subsequent translation thereby contributing to the regulation of important cellular processes. The aim of our studies in keratinocytes is to identify SM-evoked alterations in the expression of miRNAs with relevance in wound healing that may be counterbalanced by use of synthetic miRNAs (mimics) and miRNA inhibitors (anti-miRs) in order to rescue dysregulated cell functions. In the first part of our project, primary human keratinocytes are subjected to analysis by next-generation-sequencing technology aiming to compile a comprehensive signature of miRNAs and mRNAs regulated by SM. To this end, miRNA- and mRNA libraries are generated from the cells and subjected to RNA sequencing. Using specific computer software and various online databases, all miRNAs influenced by SM as well as their putative target mRNAs and potentially involved regulatory networks can be identified. A selection of the most promising miRNAs and target mRNAs are subjected to verification by experimental gene expression analysis. In the second part of our proposal, we use synthetic miRNAs (mimics) and miRNA inhibitors (anti-miRs) to investigate the role of the selected miRNAs in the control of signaling pathways important in wound healing as well as in growth, migration and differentiation of the cells. Finally, the third part of our studies is designed to validate our findings on mimic or anti-miR-based rescuing of SM-evoked cell defects obtained in cell culture by use of a complex three-dimensional epidermal skin model. The present studies allow for the first time the preparation of a comprehensive list of miRNAs and mRNAs that are regulated in keratinocytes upon exposure to SM. This may lead to the discovery and validation of novel molecular pathomechanisms, and provides experimental evidence whether mimics or anti-miRs may be basically useful as topically administrable agents for the treatment of SM-evoked wound healing disorders in the skin.

芥子气（SM）是一种剧毒战剂，在各个危机地区构成持续威胁，特别是通过恐怖主义团体的活动。皮肤暴露于SM会引起严重的组织损伤和伤口愈合缺陷，导致患者长期住院。潜在的分子和细胞病理机制研究甚少。到目前为止，没有药物治疗可用于治疗SM诱导的伤口愈合缺陷。来自皮肤细胞体外研究的结果提供了证据，表明SM影响角质形成细胞中microRNA（miRNAs）的表达和下游途径以及细胞功能。miRNA是小的普遍存在的RNA分子，其结合到特定mRNA并抑制随后的翻译，从而有助于重要细胞过程的调节。我们在角质形成细胞中的研究的目的是鉴定SM诱发的与伤口愈合相关的miRNA表达的改变，其可以通过使用合成的miRNA（模拟物）和miRNA抑制剂（抗miR）来平衡，以挽救失调的细胞功能。在我们的项目的第一部分中，原代人角质形成细胞进行分析，通过下一代测序技术，旨在编译由SM调控的miRNA和mRNA的综合签名。为此，从细胞产生miRNA和mRNA文库并进行RNA测序。使用特定的计算机软件和各种在线数据库，可以识别所有受SM影响的miRNA及其推定的靶mRNA和潜在参与的调控网络。通过实验基因表达分析对最有希望的miRNAs和靶mRNA的选择进行验证。在我们的建议的第二部分中，我们使用合成的miRNA（模拟物）和miRNA抑制剂（anti-miR）来研究所选miRNA在控制伤口愈合以及细胞生长、迁移和分化中重要的信号通路中的作用。最后，我们研究的第三部分旨在通过使用复杂的三维表皮皮肤模型来验证我们关于在细胞培养中获得的SM诱发的细胞缺陷的基于模拟或抗miR的拯救的发现。目前的研究首次允许制备暴露于SM后在角质形成细胞中受调控的miRNA和mRNA的全面列表。这可能导致新的分子病理机制的发现和验证，并提供了实验证据，无论模拟物或抗miR可以基本上作为局部给药的药物用于治疗皮肤中的SM诱发的伤口愈合障碍。