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目的探讨NEDD4-1 shRNA对U251胶质瘤细胞增殖及凋亡的影响。方法构建NEDD4-1 shRNA表达载体。以不同比例质粒和脂质体转染U251细胞,通过荧光显微镜观察细胞存活情况,优化转染效率。转染48 h后采用Western blot、RT-PCR分别检测NEDD4-1蛋白及mRNA的表达,流式细胞术检测细胞周期,MTT法检测细胞增殖,DAPI荧光染色检测细胞凋亡。结果 NEDD4-1 shRNA表达载体构建成功。脂质体与质粒的最佳转染效率比例为2.5μl∶1μg,48 h转染效率在60%～70%。与对照组比较,转染NEDD4-1 shRNA后,U251胶质瘤细胞NEDD4-1的蛋白及mRNA表达水平均下降(均P<0.05),细胞大多停滞在G0-G1期(均P<0.05),增殖率明显降低(均P<0.05),凋亡率显著增加(均P<0.05)。结论 NEDD4-1作为PTEN泛素连接酶,为胶质瘤基因治疗提供靶点。
Objective To investigate the effect of NEDD4-1 shRNA on proliferation and apoptosis of U251 glioma cells. Methods Construction of NEDD4-1 shRNA expression vector. U251 cells were transfected with plasmids and liposomes in different ratios. The cell viability was observed by fluorescence microscopy and the transfection efficiency was optimized. The expression of NEDD4-1 protein and mRNA was detected by Western blot and RT-PCR 48 h after transfection. The cell cycle was detected by flow cytometry, the cell proliferation was detected by MTT assay and the apoptosis was detected by DAPI fluorescent staining. Results NEDD4-1 shRNA expression vector was successfully constructed. The optimum transfection efficiency ratio of liposome to plasmid was 2.5 μl 1 μg, and the transfection efficiency was 60% -70% at 48 h. Compared with the control group, NEDD4-1 shRNA transfected U251 glioma cells NEDD4-1 protein and mRNA levels were decreased (P <0.05), most of the cells arrested in the G0-G1 phase (P <0.05) (All P <0.05), and the apoptosis rate was significantly increased (all P <0.05). Conclusion NEDD4-1 acts as a PTEN ubiquitin ligase and targets the gene therapy of glioma.