shRNA Notch2对人脑胶质瘤细胞株U251生长及凋亡的影响

Effects of stable transfection with shRNA-Notch2 on the proliferation,cell cycle and apoptosis of U251 glioma cell line

目的:探讨稳定转染基因Notch2短发夹状RNA(short hairpin RNA,shRNA)对胶质瘤细胞株U251生长、增殖和凋亡的影响。方法:采用脂质体法将构建插入Notch2-shRNA和无意义shRNA(Negative-shRNA)的重组表达质粒分别转染人脑胶质瘤U251细胞,建立稳定长效转染的细胞株;应用RT-PCR和蛋白质印迹法检测Notch2-shRNA对U251细胞中Notch2表达的影响及相关蛋白表达的变化;MTT法检测其细胞增殖的影响;FCM法检测细胞凋亡率和细胞周期。结果:稳定转染Notch2-shRNA后,U215细胞中Notch2 mRNA和蛋白的表达明显受到抑制;干扰组细胞从第5天开始增殖明显降低(P<0.05);干扰组细胞的凋亡率为(15.14±4.26)%,明显高于Negative-shRNA组的(2.70±1.45)%和未转染组的(2.10±1.72)%(P<0.05);Notch2-shRNA干扰组细胞S期细胞所占百分比为(23.1±3.4)%,明显低于转染Negative-shRNA组的(41.2±6.9)%和未转染组的(53.2±7.8)%(P<0.01),而干扰组G1期细胞所占百分率为(51.8±3.9)%,显著高于转染Negative-shRNA组的(38.9±9.7)%和未转染组的(25.2±7.7)%(P<0.05);与转染Negative-shRNA组和未转染组相比,干扰组细胞中p21和p27蛋白表达量上调,而细胞周期蛋白cyclinD1和微型染色体维持蛋白2(minichromosome maintenance 2 protein,MCM2)的表达量下调。结论:Notch2shRNA能有效抑制U251细胞中Notch2的表达和细胞的增殖,为脑胶质瘤基因治疗提供了新的靶点。

Objective：This study was designed to investigate the effects of stable transfection with small hairpin RNA（shRNA）-Notch2 on the proliferation,cell cycle and apoptosis of U251 glioma cell line.Methods：The shRNA expression vector which expresses the specific shRNA targeting Notch2 mRNA（Notch2-shRNA） or independent sequence（Negative-shRNA） was transfected into U251 cells,and then the U251 cells with stable expression of shRNA were selected.The expressions of Notch2 and other associated proteins were detected by RT-PCR and Western blotting.The cellular proliferation activity as well as the cell cycle and the apoptosis rate were determined by MTT assay and flow cytometry,respectively.Results：Compared with the Negative-shRNA group and the non-transfection group,the mRNA and protein expressions of Notch2 in U251 cells with stable transfection of Notch2-shRNA（interference group） was significantly suppressed and the cell proliferation was slowed down on the 5th day after transfection（P0.05）.The apoptosis rate in the interference group（15.14%±4.26%） was significantly higher than those in the Negative-shRNA group（2.70%±1.45%） and the non-transfection group（2.10%±1.72%）（P0.05）.The proportion of cells in S phase（23.1%±3.4%） was significantly lower than those in the Negative-shRNA group（41.2%±6.9%） and the non-transfection group（53.2%±7.8%）（P0.01）,whereas the proportion of cells in G1 phase（51.8%±3.9%） was significantly higher than those in the Negative-shRNA group（38.9%±9.7%） and the non-transfection group（25.2%±7.7%）（P0.05）.The expression levels of p21 and p27 proteins were down-regulated,while the cyclin D1 and minichromosome maintenance protein 2 were up-regulated in the interference group,compared with thosein the Negative-shRNA group and the non-transfection group.Conclusion：Notch2-shRNA transfection exerts long-term inhibition effects on the Notch2 mRNA expression and proliferation of U251 glioma cells,which provides a potential target in gene therapy for glioma.