Since the discovery of the first regulatory RNA strands in 1993, the number of known micro RNAs has steadily increased. However, the function of the individual molecules is often unknown. The aim of our study was to investigate the role of microRNA 154 in embryonic lung development. Our strategy included two opposing experimental approaches: A "gain of function" and a "loss of function" experiment. In both approaches, we analyzed the lung morphology and the changes at the genetic level. For the gain of function approach, we designed a mouse line that overexpressed miR-154 in the pulmonary epithelium upon doxycycline food application. We induced this process from embryonic day 7.5 until organ harvest (at E18.5). Using quantitative PCR we were able to prove the successful overexpression. As a consequence of epithelial miR-154 overexpression a phenotype with thinned alveolar septa and elongated sacculi was observed microscopically. In alveolar morphometry we found a significant reduction of septal thickness (in micrometer) and a significant increase of airspace. The parameter for volume-to-surface ratio (MLI) was also increased. Searching for the genetic correlate for this phenotype, we focused on four gene groups related to lung development: The signal cascades of the cytokines Fgf10 and Tgf-ß, as well as the marker genes for epithelial cells and alveolar myofibroblasts (AMF). AMFs are important mediators of alveolar separation. In our analysis, their marker genes showed a significant down regulation. However, the subsequent Acta2 staining did not reveal any difference in AMF presence.For the loss of function approach, we blocked miR-154 with a morpholino solution. We explanted embryonic lungs at E11.5 and cultivated the organs in vitro for 72 hours. Morphologically, the lungs of the experimental group showed a significantly reduced formation of lung buds. Gene analysis of Fgf10 and Tgf-beta signaling, but also the gene markers of epithelial cells and AMFs showed a trend towards overexpression.
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