Li Yan, Wang Jiying, Li Xiaoyan, Jia Yujiao, Huai Lei, He Kan, Yu Pei, Wang Min, Xing Haiyan, Rao Qing, Tian Zhen, Tang Kejing, Wang Jianxiang, Mi Yingchang
State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, P.R. China.
Oncol Rep. 2014 Dec;32(6):2680-6. doi: 10.3892/or.2014.3529. Epub 2014 Oct 6.
The Wilms' tumor 1 (WT1) gene is one of the regulating factors in cell proliferation and development. It is a double-functional gene: an oncogene and a tumor suppressor. This gene was found to be highly expressed in many leukemic cell lines and in patients with acute myeloid leukemia. In the present study, we demonstrated that the WT1 gene was commonly expressed in leukemic cell lines apart from U937 cells. The K562 cell line which expresses WT1 at a high level (mRNA and protein) was used in the entire experiment. By MTT and colony formation assays, we found that curcumin, an inhibitor of the WT1 protein, inhibited cell proliferation and clonogenicity in a time- and dose-dependent manner. It also caused cell cycle arrest at the G2/M phase. We then designed specific short hairpin RNAs (shRNAs) which could downregulate WT1 by 70-80% at the mRNA and protein levels. Reduction in the WT1 levels attenuated the proliferative ability and clonogenicity. Cell cycle progression analysis indicated that the proportion of cells in the G0/G1 phase increased while the proportion in the S phase decreased distinctively. ChIP-DNA selection and ligation (DSL) experiment identified a cohort of genes whose promoters are targeted by WT1. These genes were classified into different cellular signaling pathways using MAS software and included the Wnt/β-catenin pathway, MAPK signaling pathway, apoptosis pathway, and the cell cycle. We focused on the Wnt/β-catenin signaling pathway, and compared expression of several genes in the K562 cells transfected with the control shRNA and WT1-specific shRNA. β-catenin, an important gene in the Wnt canonical pathway, was downregulated after WT1 RNAi. Target genes of β-catenin which participate in cell proliferation and cell cycle regulation, such as CCND1 and MYC, were also significantly downregulated. Collectively, these data suggest that WT1 functions as an oncogene in leukemia cells, and one important mechanism is regulation of the Wnt/β-catenin pathway.
肾母细胞瘤1（WT1）基因是细胞增殖和发育的调控因子之一。它是一种双功能基因：既是癌基因又是肿瘤抑制基因。该基因在许多白血病细胞系和急性髓系白血病患者中高表达。在本研究中，我们证明WT1基因在除U937细胞外的白血病细胞系中普遍表达。在整个实验中使用了高水平表达WT1（mRNA和蛋白质）的K562细胞系。通过MTT和集落形成试验，我们发现WT1蛋白抑制剂姜黄素以时间和剂量依赖性方式抑制细胞增殖和克隆形成能力。它还导致细胞周期停滞在G2/M期。然后我们设计了特异性短发夹RNA（shRNA），其可在mRNA和蛋白质水平将WT1下调70 - 80%。WT1水平的降低减弱了增殖能力和克隆形成能力。细胞周期进程分析表明，G0/G1期细胞比例增加，而S期细胞比例明显下降。染色质免疫沉淀 - DNA选择与连接（DSL）实验鉴定出一组其启动子被WT1靶向的基因。使用MAS软件将这些基因分类到不同的细胞信号通路中，包括Wnt/β - 连环蛋白通路、MAPK信号通路、凋亡通路和细胞周期。我们聚焦于Wnt/β - 连环蛋白信号通路，并比较了用对照shRNA和WT1特异性shRNA转染的K562细胞中几个基因的表达。β - 连环蛋白是Wnt经典通路中的一个重要基因，在WT1 RNA干扰后表达下调。参与细胞增殖和细胞周期调控的β - 连环蛋白靶基因，如CCND1和MYC，也显著下调。总体而言，这些数据表明WT1在白血病细胞中作为癌基因发挥作用，一个重要机制是对Wnt/β - 连环蛋白通路的调控。
