Cells were transfected using reverse protocol and first transfection mix (100 nM miRNA precursor in 100 µl/well) and then cell suspension (400 µl with 5 x105 cells/well) were added into 24 wplates. This was followed by a spin down of the cells 200 x g for 5 minutes. Cells were incubated for 48 h before starting the treatment.

MiRNAs are a class of endogenous tiny RNAs that act as inhibitors of translation or promote RNA degradation by duplex-formation within the 3'-UTR of target mRNAs. They play an important role during a wide range of cellular processes by fine-tuning of gene expression. The differentiation of monocytes to macrophages plays a pivotal role in physiological as well as pathophysiological processes such as atherosclerosis. Monocytes which can be found in well-oxygenated blood migrate into areas with a high inflammation, such as the atherosclerotic plaque. There, they differentiate into macrophages. Interestingly, macrophages were found mainly at hypoxic sites of the plaque. Key regulators for the adaptation to hypoxia are the hypoxia-inducible factors (Hif). Therefore the aim of the present study was to investigate the regulation of the Hif-system by miRNAs during the process of monocyte differentiation. The present study shows that during the differentiation of monocytes into macrophages a dramatically change in the expression pattern of Hif-1alpha and Hif-2alpha took place. This was associated with a downregulation of microRNAs encoded by the miR-17-92 cluster. An in silico analysis of the 3'-UTR of Hif-alpha subunits for binding sites of miRNAs was performed using different miRNA databases in concert with a secondary structure prediction algorithm. This analysis revealed that both 3'-UTRs contain binding sites for miRNAs of the miR-17-92 cluster. Transfection of HeLa cells with miR-17 and miR-20a led to an inhibition of Hif-1alpha and -2alpha mRNA and protein expression and a lowered Hif DNA binding activity. Using a Luciferase-Reporter assay, it could be shown, that both Hif-alpha subunits are targeted by miR-17 and miR-20a. Furthermore, miR-overexpression in primary human macrophages demonstrates the important role of this microRNA-mediated regulation of the Hif-system for adaption of macrophages to hypoxia. In conclusion, the present study shows that the Hif-system is activated during monocyte-to-macrophage differentiation. This activation is in part mediated by a miRNA-dependent mechanism, which seems to be crucial for the adaption of macrophages to hypoxia.