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

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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.