microRNA regulated processes in keratinocytes upon exposure to sulfur mustard: modulation by mimics und anti-miRs
Cell Biology
Final Report Abstract
Mustard gas (2,2'-dichloroethyl sulfide), also known as sulfur mustard (SM), is a highly toxic warfare agent. Exposure to SM leads to serious damage, primarily to the lungs and skin. The acute, cutaneous symptoms are redness, blistering and pigmentation disorders. In the longer term, there are severe wound healing deficits. Despite intensive research, the molecular pathomechanisms of mustard-related wound healing disorders in skin cells have not yet been fully elucidated. A specific pharmacotherapy is therefore not available. Micro RNAs (miRNAs) are a group of small RNA molecules that perform regulatory tasks in cells. We suspect that exposure to SM leads to disruptions in the regulation of the production of miRNAs in the outermost skin layer (epidermis), which is formed by keratinocytes. This could impair cellular functions such as maturation (differentiation) and division (proliferation). However, these abilities are essential for the participation of keratinocytes in efficient wound healing. The aim of the present study was the genome-wide analysis of changes that SM has on the expression of mRNAs and miRNAs in keratinocytes. With the help of Next Generation Sequencing (NGS) technology, almost 30,000 different RNA molecules could be detected in the cells. In response to exposure to sulfur mustard, the production of 1,894 mRNAs and 25 miRNAs was significantly altered. The analysis of altered RNAs using special software (Gene Ontology Pathway Analysis) revealed that many are involved in cellular processes that are important for the differentiation and proliferation of keratinocytes and thus cellular function in wound healing. Experiments with four selected miRNAs, which were upregulated after treatment with SM, provided evidence that these miRNA candidates had a direct impact on the processes of differentiation and proliferation. Finally, by applying RNA- based selective inhibitors of the miRNA candidates, it was possible to eliminate the SM-induced functional defects in the keratinocytes. These findings are now to be confirmed using wound healing assays as well as human real skin biopsies. Our studies led to the discovery of a pathomechanism. This is based on the fact that under the influence of SM changes in the production of certain miRNAs occur in keratinocytes which result in a stop in cell division and trigger premature maturation of the cells. Under these conditions, keratinocytes can presumably no longer contribute adequately to wound healing. These cellular defects could be successfully counteracted in vitro by administering selective miRNA inhibitors. Our results give reason to assume that RNA-based compounds represent a promising approach for a causal therapy of mustard-related wound healing disorders.
