Kikuchi R, Murakami M, Sobue S, Iwasaki T, Hagiwara K, Takagi A, Kojima T, Asano H, Suzuki M, Banno Y, Nozawa Y, Murate T
Nagoya University Graduate School of Medicine, Nagoya University School of Health Sciences, Nagoya, Aichi, Japan.
Oncogene. 2007 Mar 15;26(12):1802-10. doi: 10.1038/sj.onc.1209973. Epub 2006 Sep 11.
It was reported that short interfering RNA (siRNA) of EWS/Fli-1 downregulated phospholipase D (PLD)2 in Ewing's sarcoma (EWS) cell line, suggesting that PLD2 is the target of aberrant transcription factor, EWS/Fli-1. Here, we further investigated the regulation of PLD2 gene expression by EWS/Fli-1 and Fli-1 in another EWS cell line, and also in EWS/Fli-1- or Fli-1-transfected cell line. EWS/Fli-1- or Fli-1-overexpressed cells showed higher PLD2 but not PLD1 protein expression and enhanced cell proliferation as compared to mock transfectant. The treatment of these cells with 1-butanol or siRNA of PLD2 inhibited cell growth, suggesting the pivotal role of PLD in cell growth promotion. PLD2 but not PLD1 mRNA level was also increased in EWS/Fli-1 or Fli-1-transfectants. After determining the transcription initiation points, we cloned the 5' promoter of both PLD1 and PLD2 and analysed promoter activities. Results showed that EWS/Fli-1 and Fli-1 increase PLD2 gene expression by binding to an erythroblast transformation-specific domain (-126 to -120 bp from the transcription initiation site) of PLD2 promoter, which is the minimal and most powerful region. Electrophoresis mobility shift assay using truncated proteins showed that both DNA-binding domain and trans-activating domain were necessary for the enhanced gene expression of PLD2.
据报道，尤文氏肉瘤（EWS）细胞系中EWS/Fli-1的小干扰RNA（siRNA）下调了磷脂酶D（PLD）2，这表明PLD2是异常转录因子EWS/Fli-1的靶点。在此，我们进一步研究了EWS/Fli-1和Fli-1对另一种EWS细胞系以及EWS/Fli-1或Fli-1转染细胞系中PLD2基因表达的调控。与mock转染细胞相比，过表达EWS/Fli-1或Fli-1的细胞显示出更高的PLD2蛋白表达，但PLD1蛋白表达未升高，且细胞增殖增强。用1-丁醇或PLD2的siRNA处理这些细胞可抑制细胞生长，这表明PLD在促进细胞生长中起关键作用。在EWS/Fli-1或Fli-1转染细胞中，PLD2而非PLD1的mRNA水平也有所增加。确定转录起始点后，我们克隆了PLD1和PLD2的5'启动子并分析了启动子活性。结果表明，EWS/Fli-1和Fli-1通过与PLD2启动子的成红细胞转化特异性结构域（转录起始位点上游-126至-120 bp）结合来增加PLD2基因表达，该结构域是最小且最强的区域。使用截短蛋白进行的电泳迁移率变动分析表明，DNA结合结构域和反式激活结构域对于增强PLD2的基因表达都是必需的。
