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Hsa-mir-145 is the top EWS-FLI1-repressed microRNA involved in a positive feedback loop in Ewing's sarcoma

Abstract

EWS-FLI1 is a chromosome translocation-derived chimeric transcription factor that has a central and rate-limiting role in the pathogenesis of Ewing's sarcoma. Although the EWS-FLI1 transcriptomic signature has been extensively characterized on the mRNA level, information on its impact on non-coding RNA expression is lacking. We have performed a genome-wide analysis of microRNAs affected by RNAi-mediated silencing of EWS-FLI1 in Ewing's sarcoma cell lines, and differentially expressed between primary Ewing's sarcoma and mesenchymal progenitor cells. Here, we report on the identification of hsa-mir-145 as the top EWS-FLI1-repressed microRNA. Upon knockdown of EWS-FLI1, hsa-mir-145 expression dramatically increases in all Ewing's sarcoma cell lines tested. Vice versa, ectopic expression of the microRNA in Ewing's sarcoma cell lines strongly reduced EWS-FLI1 protein, whereas transfection of an anti-mir to hsa-mir-145 increased the EWS-FLI1 levels. Reporter gene assays revealed that this modulation of EWS-FLI1 protein was mediated by the microRNA targeting the FLI1 3′-untranslated region. Mutual regulations of EWS-FLI1 and hsa-mir-145 were mirrored by an inverse correlation between their expression levels in four of the Ewing's sarcoma cell lines tested. Consistent with the role of EWS-FLI1 in Ewing's sarcoma growth regulation, forced hsa-mir-145 expression halted Ewing's sarcoma cell line growth. These results identify feedback regulation between EWS-FLI1 and hsa-mir-145 as an important component of the EWS-FLI1-mediated Ewing's sarcomagenesis that may open a new avenue to future microRNA-mediated therapy of this devastating malignant disease.

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Acknowledgements

We thank Aykut Uren for help in the analysis of the PDGF/mir-145 axis and Stephan Baldus for supplying primary tumor tissue, and Karin Mühlbacher for technical assistance. This study was supported by grants from the Austrian Research Fund FWF (P20665-B12) and the European Commission (EET-pipeline, LSHC-CT-2006-037260). PM is supported by the Ghent University research fund (BOF 01D31406).

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Correspondence to H Kovar.

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Ban, J., Jug, G., Mestdagh, P. et al. Hsa-mir-145 is the top EWS-FLI1-repressed microRNA involved in a positive feedback loop in Ewing's sarcoma. Oncogene 30, 2173–2180 (2011). https://doi.org/10.1038/onc.2010.581

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