Adachi Y, Matsubara S, Pedraza C, Ozawa M, Tsutsui J, Takamatsu H, Noguchi H, Akiyama T, Muramatsu T
Department of Biochemistry, Faculty of Medicine, Kagoshima University, Japan.
Oncogene. 1996 Nov 21;13(10):2197-203.
Midkine (MK) is a heparin-binding growth factor which is strongly expressed during the midgestation period of mouse embryogenesis. Wilms' tumor is an embryonal kidney malignancy in infants, and WT1 has been identified as its tumor suppressor gene. The high expression level of MK in all Wilms' tumor specimens so far examined and the presence of two WT1 elements (5'-GCGGGGGCG-3') in the human MK promoter region led us to examine the possible role of the WT1 gene product in the regulation of MK gene expression. A gel shift assay verified the complex formation between the WT1 gene product and WT1 consensus sequence of MK gene. DNase1 footprint analysis also demonstrated that the downstream WT1 element was protected from DNase1 cleavage by the addition of the WT1 protein. The human MK promoter fused with the chloramphenicol acetyltransferase gene (phMK2.3kCAT) was co-transfected with an effector plasmid containing the WT1 gene into several cell lines. Transient transfection assays showed suppression of the MK promoter by WT1 co-transfection in recipient cells; deletion of the WT1 binding site abolished the suppression. The evidence reported in this study indicates that MK gene is a newly identified WT1 target gene.
中期因子（MK）是一种肝素结合生长因子，在小鼠胚胎发育的中期强烈表达。肾母细胞瘤是婴儿期的一种胚胎性肾恶性肿瘤，WT1已被确定为其肿瘤抑制基因。迄今为止，在所有检测的肾母细胞瘤标本中MK的高表达水平以及人类MK启动子区域中两个WT1元件（5'-GCGGGGGCG-3'）的存在，促使我们研究WT1基因产物在MK基因表达调控中的可能作用。凝胶迁移试验证实了WT1基因产物与MK基因的WT1共有序列之间形成了复合物。DNase1足迹分析还表明，通过添加WT1蛋白，下游WT1元件受到保护，免受DNase1切割。将与氯霉素乙酰转移酶基因融合的人类MK启动子（phMK2.3kCAT）与含有WT1基因的效应质粒共转染到几种细胞系中。瞬时转染试验表明，在受体细胞中WT1共转染可抑制MK启动子；WT1结合位点的缺失消除了这种抑制作用。本研究报道的证据表明，MK基因是一个新发现的WT1靶基因。
