title: 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.
authors.affiliation: Children's Cancer Research Institute, St Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: J
authors.initials: J
authors.lastname: Ban
authors.forename: G
authors.initials: G
authors.lastname: Jug
authors.forename: P
authors.initials: P
authors.lastname: Mestdagh
authors.forename: R
authors.initials: R
authors.lastname: Schwentner
authors.forename: M
authors.initials: M
authors.lastname: Kauer
authors.forename: D N T
authors.initials: DN
authors.lastname: Aryee
authors.forename: K-L
authors.initials: KL
authors.lastname: Schaefer
authors.forename: F
authors.initials: F
authors.lastname: Nakatani
authors.forename: K
authors.initials: K
authors.lastname: Scotlandi
authors.forename: M
authors.initials: M
authors.lastname: Reiter
authors.forename: D
authors.initials: D
authors.lastname: Strunk
authors.forename: F
authors.initials: F
authors.lastname: Speleman
authors.forename: J
authors.initials: J
authors.lastname: Vandesompele
authors.forename: H
authors.initials: H
authors.lastname: Kovar
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics*; D034802:RNA-Binding Protein EWS / Q000235:genetics*; D012512:Sarcoma, Ewing / Q000235:genetics*
chemical_list: D017931:DNA Primers; C106031:EWS-FLI fusion protein; C541010:MIRN145 microRNA, human; D035683:MicroRNAs; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS