The role of miR-17-92 in the miRegulatory landscape of Ewing sarcoma
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Raphaela Schwentner1, David Herrero-Martin1,5, Maximilian O Kauer1, Cornelia N Mutz1, Anna M Katschnig1, Grzegorz Sienski2,6, Javier Alonso3, Dave NT Aryee1,4, Heinrich Kovar1,4
1Children´s Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna 1090, Austria
2Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna Biocenter Campus, 1030 Vienna, Austria
3Unidad de Tumores Sólidos Infantiles, Instituto de Investigación de Enfermedades Raras, ISCIII, Ctra, Majadahonda-Pozuelo Km 2, 28220 Madrid, Spain
4Department of Pediatrics, Medical University, Vienna 1090, Austria
5Present address: Sarcoma research group, Molecular Oncology Lab, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat 08908, Barcelona, Spain
6Present address: Whitehead Institute for Biomedical Research, Cambridge, MA 02142
Heinrich Kovar, email: [email protected]
Keywords: Ewing sarcoma, EWS-FLI1, miR-17-92, PAR-CLIP, TGFB/BMP pathway
Received: May 18, 2016 Accepted: December 16, 2016 Published: December 22, 2016
MicroRNAs serve to fine-tune gene expression and play an important regulatory role in tissue specific gene networks. The identification and validation of miRNA target genes in a tissue still poses a significant problem since the presence of a seed sequence in the 3′UTR of an mRNA and its expression modulation upon ectopic expression of the miRNA do not reliably predict regulation under physiological conditions. The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in Ewing sarcoma. MiR-17-92, one of the most potent oncogenic miRNAs, was recently reported to be among the top EWS-FLI1 activated miRNAs. Using a combination of AGO2 pull-down experiments by PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) and of RNAseq upon miRNA depletion by ectopic sponge expression, we aimed to identify the targetome of miR-17-92 in Ewing sarcoma. Intersecting both datasets we found an enrichment of PAR-CLIP hits for members of the miR-17-92 cluster in the 3′UTRs of genes up-regulated in response to mir-17-92 specific sponge expression. Strikingly, approximately a quarter of these genes annotate to the TGFB/BMP pathway, the majority mapping downstream of SMAD signaling. Testing for SMAD phosphorylation, we identify quiet but activatable TGFB signaling and cell autonomous activity of the BMP pathway resulting in the activation of the stemness regulatory transcriptional repressors ID1 and ID3. Taken together, our findings shed light on the complex miRegulatory landscape of Ewing Sarcoma pointing miR-17-92 as a key node connected to TGFB/BMP pathway.
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