ShRNA-AKT2对人脑胶质瘤U87细胞株增殖及凋亡的影响

Effect of stable transfection with shRNA-AKT2 on proliferation and apoptosis of U87 glioma cell line

目的研究通过慢病毒载体靶向抑制丝/苏氨酸蛋白激酶2(AKT2)基因表达对胶质瘤细胞株U87增殖、凋亡的影响。方法利用慢病毒介导的短发夹RNA(shRNA)干扰载体感染U87细胞株,逆转录酶-聚合酶链式反应(RT-PCR)和蛋白印迹技术(Western blot)分别检测转染后细胞株AKT2 mRNA和蛋白表达水平变化,四唑盐(MTT)法检测细胞增殖的影响,流式细胞术检测细胞凋亡率、细胞周期改变。结果转染AKT2-shRNA可有效降低U87细胞株内的AKT2表达。干扰组细胞从第3 d开始增殖明显降低(P<0.05);AKT2-shRNA干扰组细胞的凋亡率为11.80%±1.83%,明显高于阴性对照组的2.01%±0.20%和未转染组的2.13%±0.32%(P<0.05);AKT2-shRNA干扰组细胞周期S期细胞百分比显著低于对照组,而G0/G1期细胞比分比显著高于对照组(P<0.05)。结论 AKT2-shRNA可抑制胶质瘤细胞株的增殖,并导致肿瘤细胞凋亡增加。

Objective The effect of targeted inhibition of serine / threonine kinase β（ AKT2） gene expression on the proliferation and apoptosis of U87 cell line is studied. Methods The lentivirus vector of AKT2 short hairpin RNA（ shRNA） was constructed and transfected into U87 cells. The expressions of mRNA and protein of AKT2 were detected by reverse transcription-polymerase chain reaction（ RT-PCR） and Western blot,respectively. The cellular proliferation activity,the cell cycle and the rate of apoptosis were determined by methyl thiazolyl tetrazolium（ MTT） assay and flow cytometry,respectively. Results Compared with the negative control group and the non-transfection group,the mRNA and protein expressions of AKT2 in U87 cells with stable transfection of AKT2-shRNA（ interference group） were significantly suppressed and the cell proliferation was slowed down on the third day after the transfection（ P 0. 05）. The apoptosis rate in the interference group（ 15. 14% ± 4. 26%） was significantly higher than that in the negative control group（2. 70% ±1. 45%） and the non-transfection group（2. 10% ± 1. 72%）（P 〈0. 05）. The proportion of cells in S phase was significantly lower than that in the GFP-shRNA group and the non-transfection group（ P 0. 01）,whereas the proportion of cells in G0/ G1 phase was significantly higher than that in the negative control group and the non-transfection group（ P 〈0. 05）. Conclusion It can inhibit the proliferation of glioma cells and lead to tumor cell apoptosis by targeted inhibition of AKT2 gene expression on U87 cell line.