EZH2基因沉默对人脑胶质瘤U251细胞增殖和凋亡影响的研究

目的:研究RNA干扰技术抑制zeste基因增强子人类同源物2(EZH2)的表达对人胶质瘤U251细胞增殖及凋亡的影响.方法:构建靶向EZH2基因的shRNA质粒并转染至U251细胞中,采用RT-PCR和蛋白质印迹法检测EZH2 mRNA和蛋白的表达情况,利用四甲基偶氮哇盐(MTT)实验和Annexin V-FITC/PI流式细胞术实验观察转染后U251细胞增殖和凋亡情况.结果:靶向EZH2基因的shRNA质粒成功抑制了U251细胞EZH2基因的表达,mRNA和蛋白表达抑制率分别为56.00％和88.73％；转染shRNA EZH2后,U251细胞的生长受到明显抑制(P＜0.01),在转染96 h后,生长抑制率达35.79％；转染48 h后,U251细胞早、晚期凋亡率分别为(26.59±0.83)％和(38.63±0.80)％,较阴性质粒组和空白对照组均增加,以晚期明显,差异有统计学意义,P＜0.01.结论:EZH2基因的沉默能有效抑制胶质瘤U251细胞的增殖、促进其凋亡,提示EZH2可能成为胶质瘤基因治疗的新靶点.

Effects of EZH2 silencing on the apoptosis and proliferation of human brain glioma U251 cells

OBJECTIVE: To study the effects of EZH2 gene expression’s inhibition by RNA interference on the proliferation and apoptosis of human glioma U251 cells.METHODS: The shRNA-expressing plasmid targeting EZH2 gene was constructed and transfected into U251 cells.RT-PCR and Western blot were used to detect the EZH2 gene’s exprsssion at the level of mRNA and protein;MTT assay was performed for detecting cell proliferation and Annexin V-FITC/PI flow cytometric analysis for cell apoptosis.RESULTS: The shRNA-expressing plasmid targeting EZH2 gene successfully inhibited EZH2 gene’s expression in U251 cells,and the inhibition rates of the mRNA and protein were 56.00% and 88.73% seperately.After the transfection of the plasmid,the growth of U251 cells was signifinantly inhibited(P<0.01),and the highest growth inhibition rate was 35.79% in 96 hours.Forty-eight hours after transfection,the early and secondary apoptosis rate of U251 cells were(26.59±0.83)% and(38.63±0.80)% respectively,which were higher than both the blank control group and the negative control group(P<0.01).CONCLUSION: The silence of EZH2 gene in glioma U251 cell can inhibit its proliferation,promote its apoptosis,suggesting that EZH2 may become a new target for glioma gene therapy.