抑癌基因DKK2抑制儿童肾母细胞瘤细胞增殖的机制研究

Mechanism of tumor suppressor gene DKK2 inhibiting proliferation of nephroblastoma cells in children

目的探讨WNT信号通路调控因子DKK2在儿童肾母细胞瘤细胞和组织中的表达,及对儿童肾母细胞瘤细胞SK-NEP-1增殖的影响,并初步探讨其作用机制。方法通过RT-PCR、qRT-PCR和免疫蛋白印迹实验分析DKK2在儿童肾母细胞瘤细胞系和组织中的相对表达;将DKK2过表达的SK-NEP-1细胞设定为实验组(DKK2组),空白对照质粒组设定为对照组(Vector组),分别转染pc DNA3. 1(+)-Flag-DKK2质粒(实验组)和pc DNA3. 1(+)-Flag-Vector质粒(对照组)至SK-NEP-1细胞系,RT-PCR和免疫蛋白印迹实验验证DKK2的过表达;通过CCK-8实验和细胞克隆实验分析DKK2对细胞增殖的影响;流式细胞周期和凋亡实验验证DKK2过表达对细胞增殖的作用机制;裸鼠成瘤实验验证DKK2体外对细胞增殖的影响;通过qRT-PCR、WB及免疫组化分析DKK2对细胞增殖抑制的作用机制。结果与正常肾上皮组织相比,DKK2 mRNA在儿童肾母细胞瘤细胞和组织中的中表达明显下调,差异具有统计学意义(P <0. 001);与对照组相比(100%),转染后24、48、72 h实验组细胞活性受到明显的抑制(P <0. 05),同时实验组细胞克隆形成明显受到抑制(31. 11±2. 14)%(P <0. 05);流式细胞实验发现,与对照组相比,实验组细胞明显阻滞于G1期(P <0. 001),实验组中细胞凋亡率明显升高(P <0. 001);与对照组相比,过表达DKK2后裸鼠肿瘤和体积大小明显降低(P <0. 001);过表达DKK2后,active-β-catenin及下游的基因受到了明显抑制。结论 DKK2在人皮肤肾母细胞瘤组织中表达下调,可能通过拮抗Wnt/β-catenin信号通路参与肾母细胞瘤的机制。

Objective The aims of this study were to investigate the expression of DKK2,a WNT signaling pathway regulator,in nephroblastoma cells and tissues of children,the effect on the proliferation of nephroblastoma SK-NEP-1 cells,and to explore its mechanism.Methods The relative expression of DKK2 in nephroblastoma cells and tissues was analyzed by qRT-PCR and immunoblotting assays.Overexpressing DKK2 SK-NEP-1 cells were set as the experimental group(DKK2 group);the blank control plasmid group was set as a control group(Vector group),transfected with pcDNA3.1(+)-Flag-DKK2 plasmid(Experimental group)and pcDNA3.1(+)-Flag-Vector plasmid(Control group).The over-expression of DKK2 was confirmed in SK-NEP-1 cells by RT-PCR and immunoblotting.CCK-8 and cell cloning assays were used to determine the effect of DKK2 on cell proliferation;flow cell cycle and apoptosis assays were used to confirm the effect on cell proliferation in overexpressed DKK2 cells.The xengraft formation assay in nude mice was to verify the effect of DKK2 on proliferation in overexpressed DKK2 cells;the mechanism of DKK2 in inhibitory proliferation was analyzed by qRT-PCR,Western blotting and immunohistochemistry.Results Compared with normal renal epithelial tissues,DKK2 mRNA was down-regulated in children with nephroblastoma,and the difference was statistically significant(P<0.001).Compared with the control group,transfected DKK2 cell viability was significantly inhibited after treatment for 24,48 and 72 h(P<0.05),cell clone formation in the experimental group was significantly inhibited(31.11%±2.14%)(P<0.05),the cell cycle in the experimental group was significantly arrested at the G1 phase(P<0.001),and the apoptosis rate in the experimental group was significantly increased(P<0.001).Compared with the control group,the tumor weight and volume in nude mice were significantly low in the experimental mice which were injected DKK2 overexpression cells(P<0.001).Active-β-catenin and downstream genes were significantly inhibited in over-expressed DKK2 SK-NEP-1 cells.Conclusion DKK2 is down-regulated in human cutaneous nephroblastoma and participates in the mechanism of nephroblastoma by antagonizing Wnt/β-catenin signaling pathway.