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Schulz, Nikola (2012): microRNA profiling and target identification in a mouse model for allergic asthma. Dissertation, LMU München: Faculty of Biology
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Abstract

Allergic asthma has a high prevalence and is characterized by airway inflammation, tissue remodeling and a decline in respiratory function. Although the pathogenesis is well known, the underlying mechanisms are still poorly understood. It is believed that a fine interplay exists between the exposure to environmental stimuli and relatively small changes in expression of several genes with inter-individual variation. As microRNAs (miRNAs) are known to be responsive to environmental exposures and show dysregulated levels in diseased states, their function as regulators of gene expression might be a missing link for the changes seen in asthma. In this project, changes in miRNA expression in a mouse model for allergic asthma were investigated and the interaction with possible target genes was analyzed. Female Balb/c mice were i. p. sensitized with ovalbumin (OVA) followed by aerosol challenge on two consecutive days. An asthmatic phenotype was confirmed by elevated total cell numbers due to a rise in inflammatory cells, as well as increased CCL17 levels in broncho-alveolar lavage (BAL). High titres of OVA-specific serum IgE were measured and lung histopathology revealed infiltration of inflammatory cells with eosinophilia. To study changes in miRNA expression, whole lung RNA was subjected to miRNA-microarray analysis (Exiqon). From 580 screened miRNAs, 319 were found to be expressed, of which 36 were differentially regulated in the allergic asthma group compared to healthy control mice. A second, TaqMan® chemistry based array was performed for validation. Based on the overlap between the two arrays in addition to fold changes and p-values (Exiqon), eight miRNAs were selected for single RT-qPCR measurement. Dysregulated expression of six miRNAs could be confirmed (miRNA-21, -142-3p, -144, -205, -208, -451). Due to relatively low fold changes and in order to monitor possible co-regulation, the top 100 differentially regulated miRNAs from the Exiqon array were included in an in situ target prediction. Applying a “full consensus” approach of five prediction algorithms, 961 putative target genes were identified. Based on the assumption, that target genes harboring multiple miRNA sites might be more relevant, 11 targets containing more than four miRNA binding sites were selected. From these, the transcription factor cAMP-responsive element-binding protein 1 (CREB1) was chosen for further analysis because of its previous association with asthmatic disease. Moreover, four miRNAs (miRNA-17, -22, -144, -181a) were predicted to bind at eight different sites, one of them being miRNA-144, a significantly up-regulated miRNA identified in the initial asthma profile. To experimentally test the functional interaction between CREB1 and the predicted miRNAs, a CREB1 3´-untranslated region (UTR) containing luciferase based reporter plasmid vector was constructed and co-transfected with precursor (pre-) miRNAs into human bronchial epithelial cells. Binding of all four miRNAs could be confirmed by measuring luciferase expression. Furthermore, three of four miRNAs, when transfected alone, were able to down-regulate endogenous CREB1 expression in vitro. In the lung tissue of asthma mice, CREB1 mRNA levels were significantly reduced compared to healthy controls, in contrast to two miRNAs, miRNA-17 and -144, which showed up-regulation. To gain further insight into expression patterns during sensitization and after challenge, expression of CREB1, the two validated binding partners miRNA-17, and -144, as well as the two miRNAs (miRNA-21, -451) with most significant p-values and high fold changes from the Exiqon array were analyzed. Clear expression changes happened after OVA aerosol challenge with CREB1 levels being steadily decreased, whereas all tested miRNAs showed elevated levels at 24 h post challenge, which further intensified after 120 h. This increase resembles measurements of inflammatory cell counts in BAL pointing at a possible origin within this population. In order to test whether findings can be translated into the human situation, miRNA changes in whole blood samples of mice were compared to miRNA patterns in peripheral blood of asthmatic children. In contrast to measurements in lung tissue, all four miRNAs showed markedly decreased expression in murine blood. In human samples this reduction was mirrored and significant for miRNA-144 and -451.