敲低Aurora A基因增强替莫唑胺抗人脑胶质瘤U87细胞的作用

摘要

目的 探讨敲低AuroraA基因对替莫唑胺抗人脑胶质瘤U87细胞的作用.方法 以慢病毒介导Aurora A shRNA转染高表达AuroraA基因的U87人脑胶质瘤细胞敲低其表达;转染后荧光显微镜观察转染效率,RT-PCR、Western blot分别检测Aurora-A mRNA及蛋白表达；证实转染成功后,以不同浓度梯度替莫唑胺作用于未转染组、空载体组和转染组3组细胞,每组5个平行孔,并设空白对照及阴性对照,作用一定时间后用CCK8法测定替莫唑胺对细胞增殖影响；流式细胞仪测替莫唑胺对细胞周期的影响.结果 荧光显微镜下可见转染组及空载体组细胞带绿色荧光；未转染组、空载体组和转染组的RT-PCR和Western blot结果灰度值分别为(31023±926)、(30124±1074)、(896±172)和(39556±2306)、(39348±2738)、(574±96).相对于空载体组和未转染组,转染组的Aurora A mRNA和蛋白表达均降低(P＜ 0.001)；未转染组、空载体组和转染组的半数有效抑制浓度(IC50)分别为:(43.38±2.15) μmol/L、(43.76±1.52)μmol/L、(22.20±2.34) μmol/L,转染组替莫唑胺半数有效抑制浓度(IG0)降低(P＜0.01)；替莫唑胺处理的转染组G2/M期细胞百分数(88.01％±7.35％)高于空载体组(59.28％±6.76％)和未转染组(58.35％±5.98)(P＜ 0.01),与对照的相同细胞株相比GVM期均延长(P＜0.01).结论 敲低AuroraA基因表达可增强替莫唑胺抗人脑胶质瘤U87细胞作用.%Objective To investigate the inhibitory effect of combination of Aurora A gene knockdown and Tomozolomide on growth of U87 glioma cells. Method A U87 glioma cell line with a high level of Aurora A expression was transfected with lentiviruses encoding an Aurora A shRNA. Fluorescence microscopy, RT-PCR and Western blot were used to assess cell transfection, Aurora A mRNA and protein expression, respectively. Different concentrations of Tomozolomide were administered to transfected group, empty vector group and non-transfected group (5 wells per each group). CCK8 assay and Flow cytometry were used to detect cell proliferation, and cell apoptosis as well as cell cycle, respectively. Results Fluorescence microscopy demonstrated that green fluorescence was evident in transfected group and empty vector group. The average intensity of Aurora A in RT-PCR was 31023 ± 926 in non-transfected group, 30124 ± 1074 in empty vector group and 896 ± 172 in transfected group, respectively. The average band intensity of Aurora A in Western blot was 39556 ± 2306 in non-transfected group, 39348 ± 2738 in empty vector group and 574 ± 96 in transfected group, respectively. Compared with empty vector and non-transfected groups, the mRNA and protein levels of Aurora A were reduced in transfected group (P < 0.001). The IC50 for inhibition of Aurora was 43.38 ± 2.15 μmol/L in non-trasnfected group, 43.76 ± 1.52 μmol/L in empty vector group and 22.20 ± 2.34 μmol/L in transfected group, respectively. The IC50 of inhibition of tomozolomide on Aurora A significantly rndecreased in transfeeted group (P < 0.01). The percentage of G2/M cell number was higher in transfected group (88.01% ± 7.35%) than in empty vector group (59.28% ± 6.76%) and non-transfected group (58.35% ± 5.98) (P < 0.01). compared with control group, the combination of Aurora A gene knockdown and Tomozolomide significantly prolonged the G2/M cell cycle (P < 0.01). Conclusion These results indicate that combination of Aurora A gene knockdown and Tomozolomide enhances inhibition of glioma cell growth. Aurora A gene may be a target for gene therapy of glioma.