Abstract
Ewing sarcoma, the second most common bone tumor in children and young adults, is an aggressive malignancy with a strong potential to metastasize. Ewing sarcoma is characterised by translocations encoding fusion transcription factors with an EWSR1 transactivation domain fused to an ETS family DNA binding domain. microRNAs are post-transcriptional regulators of gene expression and aberrantly expressed microRNAs have been identified as tumor suppressors or oncogenes in most cancer types. To identify potential oncogenic and tumor suppressor microRNAs in Ewing sarcoma, we determined and compared the expression of 377 microRNAs in 40 Ewing sarcoma biopsies, 6 Ewing sarcoma cell lines and mesenchymal stem cells, the putative cellular origin of Ewing sarcoma, from 6 healthy donors. Of the 35 differentially expressed microRNAs identified (fold change >4 and q<0.05), 19 were higher and 16 lower expressed in Ewing sarcoma. In comparisons between Ewing sarcoma samples with EWS-FLI or EWS-ERG translocations, with differing dissemination characteristics and of primary samples and metastases no significantly differential expressed microRNAs were detected using various stringency criteria. For miR-31, the microRNA with lowest expression in comparison to mesenchymal stem cells, functional analyses were performed to determine its potential as a tumor suppressor in Ewing sarcoma. Two of four miR-31 transfected Ewing sarcoma cell lines showed a significantly reduced proliferation (19% and 33% reduction) due to increased apoptosis in one and increased length of G1-phase in the other cell line. All three tested miR-31 transfected Ewing sarcoma cell lines showed significantly reduced invasiveness (56% to 71% reduction). In summary, we identified 35 microRNAs differentially expressed in Ewing sarcoma and demonstrate that miR-31 affects proliferation and invasion of Ewing sarcoma cell lines in ex vivo assays.
Highlights
Ewing sarcoma (ES) is the second most frequent bone tumor in children and young adults with an overall incidence of about 1.3 cases per million people [1,2]
This indicated a consistently differential miRNA expression in ES samples compared to mesenchymal stem cells (MSCs), and consistent differences in miRNA expression in ES cell lines compared to ES biopsies, which were most likely due to the adaption of the ES cell lines to cell culture conditions
For the remaining 28 miRNAs with differential expression in ES biopsies but not in ES cell lines, it could not be excluded that their differential expression in ES biopsies was due to cell culture adaption of the in-vitro expanded MSCs used for comparison and these miRNAs were excluded from further analyses
Summary
Ewing sarcoma (ES) is the second most frequent bone tumor in children and young adults with an overall incidence of about 1.3 cases per million people [1,2]. The typical genomic aberration in ES is a translocation between the EWSR1 gene and an ETS-family member with FLI1 in 85% and ERG in 5–10% of cases. In the resulting fusion protein the transactivation domain of EWS is combined with the DNAbinding domains of FLI1 or ERG to create an aberrant transcription factor [6,7], which effects the expression of more than 1000 genes [8,9]. Most evidence indicates that mesenchymal stem cells (MSCs) are the progenitors of ES. EWSFLI can induce malignant transformation of murine MSCs, it is by itself insufficient to transform human stem cells indicating that other cooperating events are required [11,13]
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