Knockdown of EWSR1/FLI1 expression alters the transcriptome of Ewing sarcoma cells in vitro

Abstract

Ewing sarcoma breakpoint region 1 (EWSR1) fusion with Friend leukemia integration 1 transcription factor (FLI1) induced by a translocation of chromosome 11 with 22 contributes to Ewing sarcoma development. To date, the precise molecular mechanisms about EWSR1/FLI1 involving in Ewing sarcoma development remains to be defined. This study explored the potential critical gene targets of EWSR1/FLI1 knockdown in Ewing sarcoma cells on the gene expression profile based on online dataset, performed Limma algorithm for differentially expressed genes identification, constructed the transcriptional factor (TF)-gene regulatory network based on integrate transcriptional regulatory element database (TRED). The data showed up- and down-regulation of differentially expressed genes over time and peaked at 72 h after EWSR1/FLI1 knockdown in Ewing sarcoma cells. SMAD3 were up-regulated and FLI1, MYB, E2F1, ETS2, WT1 were down-regulated with more than half of their targets were down-regulated after EWSR1/FLI1 knockdown. The Gene Ontology (GO) and pathway annotation of these differentially expressed genes showed a consistent trend in each group of samples. Totally, there were 355 differentially expressed genes occurring in all five comparison groups of different time points, in which 39 genes constructed a dysregulated TF-gene network in Ewing sarcoma cell line A673 after EWSR1/FLI1 knockdown. These data demonstrated that knockdown of EWSR1/FLI1 expression led to transcriptome changes in Ewing sarcoma cells and that Ewing sarcoma development and progression caused by altered EWSR1/FLI1 expression may be associated with more complex transcriptome changes.

Keywords: Ewing sarcoma; Ewsr1/fli1 fusion protein; Transcriptional factors-gene regulatory network; Transcriptome.

Fig. 1

Differentially expressed genes after EWSR1/FLI1 knockdown in Ewing sarcoma cells. The gene expression datasets GSE27524 were downloaded from GEO Datasets of NCBI (http://www.ncbi.nlm.nih.gov/gds/). The datasets GSE27524 contained a cDNA microarray analysis of A673 in Ewing sarcoma cell line after inducible EWSR1/FLI1 knockdown with up to 96 h data . We performed the limma algorithm in R/Bioconductor for genes with a p-value <0.05 and fold change >2 to considered as DEGs compared to the 0 h control.

Fig. 2

Summary of GO and KEGG terms of these DEGs among these five time-points vs. oh control in Ewing sarcoma cells after EWSR1/FLI1 knockdown. These DEGs were subjected to the Gene Ontology and KEGG pathway analysis.

Fig. 3

Construction of the TF-gene regulatory network and bi-clusters analysis of these DEGs. (A) The regulation information are derived from TRED. Triangles are TFs (orange for up-regulated, yellow for down-regulated); circles are target genes (red for up-regulated genes and green circles for down-regulated genes). The direction of arrows is from the Source (TFs) to the Target. (B) The Bi-cluster analysis of all DEGs in the TF-gene regulatory network based on gene expression value in each sample. Each row represents a gene and each column represents a sample.