RNA干扰敲低Wnt2的表达抑制U251细胞生长的体内外研究

The suppressive effect of knocking down the Wnt2 expression on glioma cells by RNAi ： an in vitro and in vivo study

目的 在体内外研究转染靶向Wnt2的siRNA对U251细胞的抑制效果.方法 向U251细胞转染靶向Wnt2的siRNA后,免疫印记检测转染后Wnt2及相关蛋白表达,MTT检测细胞增殖,annexin标记细胞凋亡,流式细胞仪检测细胞周期分布,鼠尾胶3D生长实验检测细胞侵袭能力的变化.建立裸鼠胶质瘤皮下动物模型,瘤内注射Wnt2 siRNA观察肿瘤生长情况.结果 转染Wnt2siRNA后,U251细胞的Wnt2、frizzled2、磷酸化GSK-3β、β-catenin等表达降低,细胞增殖受抑,凋亡率升高,细胞阻滞于G0/G1期.体内研究发现转染靶向Wnt2的siRNA后,裸鼠皮下肿瘤的生长速度缓慢,相应的蛋白表达降低.结论 转染靶向Wnt2的siRNA可有效敲低该基因在U251细胞内的表达,从而抑制了胶质瘤细胞生长,具有潜在的治疗前景.

Objective To study the suppressive effect of knocking down Wnt2 expression by RNAi on U251 glioblastoma cell growth in vitro and in vivo.Method siRNA targeting Wnt2 was transfected into U251 cells and the expression of Wnt2,β-catenin and main components in Wnt pathway was studied by Western blotting and immunofluorescence staining.The phenotypic change of U251 cells including proliferation,apoptosis and invasive ability after Wnt2 siRNA transfection was determined by MTT assay,annexin V staining,flow cytometry and Matrigel 3D growth experiment.Subcutaneous U251 gliomas in nude mice were treated with Wnt2 siRNA.The tumor growth was observed and compared with untreated mice with xenograft tumors.Results Aftor transfection with wnt2 siRNAa,the expresston of Wnt2,frizzled2,phosphorylated GSK-3β and,β-catenin protein was decreased.U251 cells transfected with Wnt2 siRNA showed lowering proliferation activity,decrease of SPF and arresting cell cycle in G0/G1 phase.The cell invasive ability was attenuated and cell apoptosis was induced.Subcutaneous U251 gliomas in nude mice treated with siRNA targeting Wnt2 grew slowly and the tumor volume was much smaller than that in control group.Conclusions Using RNAi technology,siRNA targeting Wnt2 efficiently knocks down the expression of Wnt2 in human glioma cells,inhibits activation of Wnt signaling pathway,results in suppression of tumor cell growth.Wnt2 siRNA is a potential therapeutic target for malignant gliomas.