Marques Howarth Michelle, Simpson David, Ngok Siu P, Nieves Bethsaida, Chen Ron, Siprashvili Zurab, Vaka Dedeepya, Breese Marcus R, Crompton Brian D, Alexe Gabriela, Hawkins Doug S, Jacobson Damon, Brunner Alayne L, West Robert, Mora Jaume, Stegmaier Kimberly, Khavari Paul, Sweet-Cordero E Alejandro
J Clin Invest. 2014 Dec;124(12):5275-90. doi: 10.1172/JCI72124. Epub 2014 Nov 17.
Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma-associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes.
导致编码RNA结合蛋白EWS和转录因子FLI1（EWS-FLI1）的基因融合的染色体易位是尤因肉瘤的特征性表现。EWS-FLI1通过尚未完全了解的机制改变基因表达。我们对表达EWS-FLI1的原发性小儿人间充质祖细胞（pMPC）进行了RNA测序（RNAseq）分析，以鉴定这种癌蛋白的基因靶点。我们确定长链非编码RNA-277（尤因肉瘤相关转录本1 [EWSAT1]）在pMPC中被EWS-FLI1上调。抑制EWSAT1表达会降低尤因肉瘤细胞系在软琼脂中增殖和形成集落的能力，而抑制EWSAT1对其他测试细胞类型没有影响。EWS-FLI1和EWSAT1的表达抑制基因表达，并且很大一部分被EWS-FLI1抑制的靶点也被EWSAT1抑制。对原发性人类尤因肉瘤的RNAseq数据的分析进一步支持了EWSAT1在介导基因抑制中的作用。我们鉴定出异质性核核糖核蛋白（HNRNPK）作为一种与EWSAT1相互作用的RNA结合蛋白，并发现HNRNPK抑制的基因与EWS-FLI1和EWSAT1抑制的基因有明显重叠，这表明HNRNPK参与了EWSAT1介导的基因抑制。总之，我们的数据表明EWSAT1是EWS-FLI1的下游靶点，它通过抑制靶基因促进尤因肉瘤的发展。
