Abstract 3080: Definition of the Ewing sarcoma specific oncomir-1 targetome

Abstract

Abstract MicroRNAs (miRNA) serve to fine-tune gene expression and thus play an important regulatory role in tissue specific gene networks. The identification and validation of miRNA target genes in a given 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 forced ectopic expression of the miRNA do not reliably predict regulation under physiological conditions. Here, we used a combination of AGO2 pull-down experiments by PAR-CLIP - to enrich and sequence RISC-associated RNAs - and of RNAseq upon miRNA depletion by ectopic sponge expression, to identify the targetome of miR17-92 in Ewing sarcoma (ES) cells. The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in ES. As an aberrant ETS transcription factor it directly deregulates a multitude of genes related to proliferation control and differentiation. Recently, we reported on the EWS-FLI1 microRNA (miRNA) signature based on knockdown experiments in ES cell lines and on differential expression in primary tumors versus mesenchymal stem cells. Comparison of both datasets revealed miR20a-3p to be the top EWS-FLI1 activated miRNA. MiR-20a belongs to the miR17-92 cluster, also known as oncomir-1, which is one of the most potent oncogenic miRNAs. The whole cluster is among the small number of miRNAs down-regulated upon knockdown of EWS-FLI1 in multiple ES cell lines. Since feedback loops exists between the E2F transcription factor family and miR-17-92 and between EWS-FLI1 and E2F3, we sought to investigate the role of this miRNA cluster in ES. We found a significant 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. Among them we consistently identified known (i.e. CTGF, MXD1, CYLD1) and a multitude of so far unknown targets of the oncomir-1 cluster in ES in three PAR-CLIP experiments. Preliminary experiments using 3′UTRs of select novel candidate genes in gene reporter assays showed up-regulation of luciferase activity upon transfection of a sponge specific to miR-17-92 and 20a, but not to unrelated miR-9 and 10b. Site directed mutagenesis of seed sequences in the 3′UTR of these candidate genes will be used for further validation. This study provides an example for the comprehensive definition of the targetome for a defined group of miRNAs under physiological conditions. This study was supported by the 7th framework program of the European Commission (FP7-259348, “ASSET”) and the Austrian Science Fund FWF (24708-B21) Citation Format: Raphaela Schwentner, David Herrero-Martin, Maximilian Kauer, Heinrich Kovar. Definition of the Ewing sarcoma specific oncomir-1 targetome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3080. doi:10.1158/1538-7445.AM2015-3080