小干扰RNA介导神经纤毛蛋白-1对西地尼布治疗脑胶质细胞瘤细胞学功能的影响

Effects of neuropilin-1 mediated by siRNA on cytological function of glioma treated with cediranib

目的探讨小干扰RNA(siRNA)介导神经纤毛蛋白-1(NRP-1)对西地尼布(Ced)治疗脑胶质细胞瘤细胞学功能的影响,为脑胶质细胞瘤患者的个体化诊疗提供依据。方法选取3种恶性程度不同的人脑胶质细胞瘤细胞系U87、U251和H4进行研究。用细胞计数法(CCK-8)检测4个不同浓度梯度(0,3,6,9 mg·mL^(-1))西地尼布处理后3种胶质细胞瘤细胞系增殖能力以筛选其最适治疗浓度。本研究共3种细胞类型,每种细胞类型分为4组。NRP-1-siRNA组:NRP-1-siRNA序列分别转染脑胶质瘤细胞; Ced-NRP-1-siRNA联合组:在NRP-1-siRNA组基础上加入最适治疗浓度西地尼布;西地尼布组:最适治疗浓度西地尼布处理;空白对照组:未做任何处理。用CCK-8法和流式细胞仪分别检测各组细胞增殖和凋亡能力;用实时荧光定量PCR和酶联免疫吸附法检测各组细胞中NRP-1mRNA和血管内皮细胞生长因子受体-1(VEGFR-1)蛋白表达水平。结果 U87细胞的空白对照组、西地尼布组、NRP-1-siRNA组和Ced-NRP-1-siRNA联合组在第4天细胞凋亡率分别为(6. 46±0. 56)%,(10. 81±1. 19)%,(20. 16±1. 89)%和(32. 48±1. 77)%;U251细胞的空白对照组、Ced组、NRP-1-siRNA组和Ced-NRP-1-siRNA联合组在第4天细胞凋亡率分别为(9. 12±0. 62)%,(13. 68±1. 08)%,(15. 45±0. 75)%和(22. 34±1. 64)%;与U251细胞比较,U87细胞各组细胞凋亡率明显下降,差异有统计学意义(P <0. 05)。与U251细胞比较,U87细胞各组细胞NRP-1 mRNA和VEGFR-1蛋白表达水平增高,差异均有统计学意义(均P <0. 05)。结论 NRP-1在U87中表达水平高于U251和H4,靶向敲除其可增强西地尼布治疗脑胶质瘤疗效,以高恶性U87更为明显,为脑胶质瘤的个体化靶向治疗奠定了基础。

Objective To explore the effects of neuropilin-1(NRP-1)mediated by small interfering RNA(siRNA)on cytological function of glioma treated with cediranib(Ced)to provide basis for individualized diagnosis and treatment of patients with glioma.Methods Three different malignant glioma cell lines U87,U251 and H4 were selected for study.The CCK-8 assay was used to determine the proliferative capacity of three glioma cell lines treated with different concentration gradients(0,3,6 and 9 mg·mL-1)Ced to screen the optimal therapeutic concentration.There were three cell types in this study,each cell type was divided into four groups:NRP-1-siRNA group:NRP-1-siRNA sequence was transfected into glioma cells;Ced-NRP-1-siRNA combined group:on the basis of NRP-1-siRNA group,the optimal treatment concentration Ced was added;Ced group:the optimal treatment concentration Ced treatment;blank control group:no treatment.The cell proliferation and apoptosis were detected by CCK-8 and flow cytometry respectively.The expression levels of NRP-1 mRNA and vascular endothelial growth factor receptor-1 were detected by real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay.Results The apoptotic rates of U87 cells in blank control group,Ced group,NRP-1-siRNA group and Ced-NRP-1-siRNA combination group were(6.46±0.56)%,(10.81±1.19)%,(20.16±1.89)%and(32.48±1.77)%on the 4 th day;and the apoptotic rates of U251 cells in blank control group,Ced group,NRP-1-siRNA group and Ced-NRP-1-siRNA combination group were(9.12±0.62)%,(13.68±1.08)%,(15.45±0.75)%and(22.34±1.64)%on the 4 th day;compared with U251 cells,the apoptotic rate of U87 cells decreased significantly,and the difference was statistically significant(P<0.05).Compared with U251 cells,the expression levels of NRP-1 mRNA and VEGFR-1 protein in U87 cells increased significantly,and the difference was statistically significant(all P<0.05).Conclusion The expression level of NRP-1 in U87 is higher than that of U251 and H4,targeted knockout can enhance the efficacy of cediranib in the treatment of glioma,especially in high malignant U87,which lays the foundation for individualized targeted therapy of glioma.