mir-34a模拟物转染人脑胶质瘤细胞U87对Dll1基因表达的影响

Effect of Mir-34a Mimics Transfected Human Brain Glioma Cell Line U87 on Expression of Dll1 Gene

目的观察mir-34a对人脑胶质瘤细胞U87中Dll1基因和蛋白表达水平的影响,探讨Dll1是否为mir-34a的靶基因。方法将人脑胶质瘤U87细胞分为mir-34a模拟物组、阴性对照组、脂质体组和空白对照组,根据人源mir-34a序列,设计并合成其双链模拟物(mir-34a mimics)。将mir-34a mimics以脂质体Lipofectamine 2000包裹,并转染至人脑胶质瘤细胞U87中,转染后48 h采用实时荧光定量PCR方法检测细胞内Dll1基因mRNA表达水平,Western blot技术检测细胞内Dll1蛋白表达水平。实验数据采用单因素方差分析和t检验进行统计学分析。结果转染后48 h模拟物组、阴性对照组、脂质体组和空白对照组细胞内Dll1基因mRNA相对表达水平分别为1.26±0.09、1.29±0.03、1.10±0.12和1.39±0.08;转染后48 h模拟物组、阴性对照组、脂质体组和空白对照组细胞内Dll1蛋白相对表达水平分别为0.011 34±0.041 90、0.628 09±0.035 00、0.913 28±0.036 20和0.868 60±0.039 00。模拟物组Dll1蛋白表达水平均明显低于阴性对照组、脂质体组和空白对照组(Pa<0.05);而模拟物组Dll1的mRNA表达水平与其他3组比较差异均无统计学意义(Pa>0.05)。结论人脑胶质瘤细胞U87中,mir-34a可通过下调Dll1蛋白而非基因表达水平发挥该靶基因转录后翻译水平的负性调控作用。

Objective To investigate the effect of mir-34a on Dll1 mRNA and protein expressions in human glioma cell line U87 and determine whether Dll1 gene was the target of mir-34a.Methods The human glioma U87 cells were divided into mir-34a mimics group,negative control group,lipofectamine group and blank control group.According to human mir-34a sequences,double-stranded mimics of mir-34a were designed and transfected into U87 cells with Lipofectamine 2000.After transfection for 48 h,total RNA and protein were extracted,and real-time fluorescent quantitative PCR was used to detect the expression of Dll1 mRNA while Western blot was used to test protein expression of Dll1.All data were analyzed with One-Way ANOVA and t test.Results Dll1 mRNA expressions in 4 groups at 48 h after transfection were 1.26±0.09,1.29±0.03,1.10±0.12 and 1.39±0.08,respectively.Dll1 protein levels in these groups at 48 h after transfection were 0.011 34±0.041 90,0.628 09±0.035 00,0.913 28±0.036 20 and 0.868 60±0.039 00,respectively.Compared with 3 control groups,the expression level of Dll1 protein in mir-34a mimics group was obviously down-regulated（Pa0.05）.However,there was no significant difference in mRNA expression level between these groups（Pa0.05）.Conclusions In U87 cell,mir-34a plays a ne-gative role in the regulation of Dll1 target gene through down-regulating Dll1 protein but not mRNA.