In this study, we explored the regulation and the role of up-regulated microRNAs in idiopathic pulmonary fibrosis (IPF), a progressive interstitial lung disease of unknown origin. We analyzed the expression of microRNAs in IPF lungs and identified 43 significantly upregulated microRNAs. Twenty-four of the 43 increased microRNAs were localized to the chromosome 14q32 microRNA cluster. We validated the increased expression of miR-154, miR-134, miR-299-5p, miR-410, miR-382, miR-409-3p, miR-487b, miR-31, and miR-127 by quantitative RT-PCR and determined that they were similarly expressed in embryonic lungs. We did not find evidence for differential methylation in this region, but analysis of transcription factor binding sites identified multiple SMAD3-binding elements in the 14q32 microRNA cluster. TGF-b1 stimulation of normal human lung fibroblasts (NHLF) caused up-regulation of microRNAs on chr14q32 that were also increased in IPF lungs. Chromatin immunoprecipitation confirmed binding of SMAD3 to the putative promoter of miR-154. Mir-154 was increased in IPF fibroblasts, and transfection of NHLF with miR-154 caused significant increases in cell proliferation and migration. The increase in proliferation induced by TGF-b was not observed when NHLF or IPF fibroblasts were transfected with a mir-154 inhibitor. Transfection with miR-154 caused activation of the WNT pathway in NHLF. ICG-001 and XAV939, inhibitors of the WNT/b-catenin pathway, reduced the proliferative effect of miR-154. The potential role of miR-154, one of multiple chr14q32 micro-RNA cluster members up-regulated in IPF and a regulator of fibroblast migration and proliferation, should be further explored in IPF.

Motivation: Pairing between the target sequence and the 6–8 nt long seed sequence of the miRNA presents the most important feature for miRNA target site prediction. Novel high-throughput technologies such as Argonaute HITS-CLIP afford meanwhile a detailed study of miRNA:mRNA duplices. These interaction maps enable a first discrimination between functional and non-functional target sites in a bulky fashion. Prediction algorithms apply different seed paradigms to identify miRNA target sites. Therefore, a quantitative assessment of miRNA target site prediction is of major interest.Results: We identified a set of canonical seed types based on a transcriptome wide analysis of experimentally verified functional target sites. We confirmed the specificity of long seeds but we found that the majority of functional target sites are formed by less specific seeds of only 6 nt indicating a crucial role of this type. A substantial fraction of genuine target sites arenon-conserved. Moreover, the majority of functional sites remain uncovered by common prediction methods.Contact: florian.buettner@helmholtz-muenchen.de v.stuempflen@helmholtz-muenchen.deSupplementary Information: Supplementary data are available at Bioinformatics online.