Genetic alterations in nephroblastomas and anti-proliferating effects induced by silencing of the tumor-related genes.

为探讨β-连环素基因突变在肾母细胞瘤发生中的作用，将正常的β-连环素基因或不同类型的突变序列导入胚胎肾来源的HEK293细胞，建立稳定的克隆。选择了三个不同的克隆，分别对应于正常的β-连环蛋白序列(WT)、密码子45的点突变(P45)和β-连环蛋白基因的整个外显子3的间隙缺失(ΔEX3)。在这些细胞系中，引入的β-连环素的外源表达受到多西环素(DOX)的调节。随后利用TOPFLASH质粒系统对转录活性的分析表明，在WT和P45细胞系中，外源过表达的β-连环蛋白通过T细胞因子结合位点增强了报告基因的转录，但在ΔEX3中没有。然而，进一步的研究未能显示这三种细胞系中的任何一种细胞增殖增加，没有明确的证据表明β-连环素异常参与了肾母细胞瘤的发展。另一方面，在神经母细胞瘤细胞系NB-1上的MYCN基因沉默实验显示，细胞增殖能力受到抑制，并伴随着细胞的凋亡和分化。由于MYCN基因在NB-1中的扩增和过度表达类似于许多其他临床肿瘤，因此抑制该基因的策略可能会带来一种新的有希望的治疗方法

To investigate how much extent beta-catenin aberration would have positive effect on tumorigenesis of nephroblastomas, normal cDNA of beta-catenin, or various types of mutated sequences were introduced into HEK293 cells derived from the embryonal kidney, and stable clones were established. Three different clones, corresponding to the normal beta-catenin sequence (WT), a point mutation at codon 45 (P45), and an interstitial deletion of the whole exon 3 of the bete-catenin gene (Δ ex3), were selected. Exogenous expressions of the introduced beta-catenins were regulated by administration of doxycycline (DOX) in these cell lines. Subsequent analyses of transcriptional activity using TOPFLASH plasmid system showed that exogenously overexpressed beta-catenins enhanced transcription of the reporter gene regulated through T-cell factor binding site in the WT and P45 cell lines, but not in the Δ ex3. Further investigation, however, failed to show increased cell proliferation in any of these three cell lines, providing no definitive supports to indicate involvement of beta-catenin aberration in the development of nephroblastic tumors.On the other hand, MYCN gene silencing experiment on a neuroblastoma cell line, NB-1, demonstrated suppressed cell proliferation property accompanied by apoptosis and differentiation of the cells. Because the MYCN gene is amplified and overexpressed in NB-1 similar to many other clinical tumors, strategy to silence this gene may lead to a new hopeful therapy