Abstract: 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.

Key words: Nephroblastoma, DKK2, Tumor suppressor genes, Cell proliferation, Wnt/β-catenin