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

硫磺芥子(SM)是一种剧毒的战剂，在各种危机地区构成持续威胁，特别是通过恐怖组织的活动。暴露在SM的皮肤会引起严重的组织损伤和伤口愈合中的缺陷，导致患者长期住院。其潜在的分子和细胞致病机制研究较少。到目前为止，还没有有效的药物治疗方法来治疗SM诱导的Will愈合缺陷。自己在皮肤细胞中的体外研究结果提供了证据，表明SM影响角质形成细胞中microRNAs(MiRNAs)的表达及其下游途径和细胞功能。MiRNAs是一种普遍存在的小的RNA分子，它与特定的mRNAs结合并抑制随后的翻译，从而有助于调节重要的细胞过程。我们在角质形成细胞中的研究目的是确定SM诱导的miRNAs表达的变化与伤口愈合有关，这种变化可以通过使用合成的miRNAs(模拟物)和miRNA抑制剂(抗miRs)来抵消，以挽救失调的细胞功能。在我们项目的第一部分，我们对原代人类角质形成细胞进行了分析-下一代测序技术，旨在编制一个全面的miRNAs和受SM调控的mRNAs的签名。为此，从细胞中生成miRNA-和mRNA文库，并进行RNA测序。利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miRNAs以及它们可能的靶标mRNAs和可能涉及的调控网络。最有希望的miRNAs和靶mRNAs的选择通过实验基因表达分析进行了验证。在我们的建议的第二部分，我们使用合成的miRNAs(模拟物)和miRNA抑制剂(抗miRs)来研究选定的miRNAs在控制创伤愈合以及细胞生长、迁移和分化中的重要信号通路中的作用。最后，我们研究的第三部分旨在验证我们的发现，即通过使用复杂的三维表皮皮肤模型来模拟或反miR修复细胞培养中获得的SM诱发的细胞缺陷。本研究首次提供了在SM作用下角质形成细胞中受调控的miRNAs和mRNAs的综合清单。这可能导致新的分子病理机制的发现和验证，并为仿制药或抗miRs作为局部用药治疗SM诱发的皮肤创伤愈合障碍提供实验证据。

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.