RNA干扰沉默DDX1基因的胶质瘤细胞系的建立

Establishment of stable glioma cell line with DDX1 gene silencing by RNA interference

目的以短发夹核糖核酸(RNA)慢病毒载体建立DEAD-box解旋酶1(DDX1)基因稳定沉默的胶质母细胞瘤细胞株。方法针对DDX1基因设计2组短发夹RNA序列,退火形成的双链DNA与线性p LKO. 1-puro-e GFP质粒载体连接,构建慢病毒质粒载体,测序正确后进行慢病毒包装;将获得的重组慢病毒转染人胶质瘤U251细胞,转染细胞分为对照组和干扰组,对照组转染对照慢病毒(sh NC);干扰组转染慢病毒载体p LKO. 1-puro-e GFP-shRNA-DDX1(sh DDX1-1,sh DDX1-2)。用荧光显微镜观察绿色荧光蛋白表达,进行嘌呤霉素抗性筛选;用实时定量聚合酶链式反应(PCR)、蛋白免疫印迹法检测转染后U251细胞中DDX1 mRNA及蛋白表达。结果测序证实成功构建对照组和靶向DDX1基因的RNA干扰(RNAi)慢病毒载体,病毒悬液滴度均> 3×10~8TU·m L^(-1)。荧光显微镜下观察显示,对照组和干扰组转染效率高于85%;嘌呤霉素筛选后,干扰组sh DDX1-1、sh DDX1-2中DDX1 mRNA的抑制率分别为85. 44%和71. 66%,DDX1蛋白表达水平分别下降了99. 1%和96. 7%,均较对照组(100%)显著降低(均P <0. 01)。结论成功构建沉默DDX 1基因的胶质瘤细胞系,为研究DDX1对胶质瘤生物学行为的影响提供细胞模型。

Objective To establish stable glioblastoma cell line with DEAD-box helicase 1 (DDX1) gene silencing by short hairpin ribonucleic acid (RNA) lentiviral vector.Methods Two sets of short hairpin RNA targeting DDX1 gene were designed.The double-stranded DNA by annealing was ligated with a linear pLK0.1-puro-eGFP plasmid to construct a lentiviral vector,which was used for lentiviral packaging subsequently. Human glioma U251 cells were infected with lentivirus.The infected cells were divided into two groups: control group,infected with control lentivirus (shNC) ; interference group,infected with virus containing lentiviral vector pLK0.1-puro-eGFP-shRNA-DDX1 (shDDX1-1,shDDX1-2) .After transfection,the expression of green fluorescence protein (GFP) was observed under fluorescence microscope; meanwhile puromycin selection for the positive infected cells was performed.The expression level of DDX1 mRNA and protein in U251 cells was detected by real-time quantitative PCR and western blot,respectively. Results The successful construction of RNA interference (RNAi) lentiviral vectors targeting negative control and DDX1 gene were confirmed by sequencing.The titer of the virus suspension was more than 3×10^8 TU·mL^-1.The transfection efficiency was higher than 85%,which was indicated by observation of GFP expression under fluorescence microscope.After puromycin selection,the inhibitory rates of DDX1 mRNA in shDDX1-1,shDDX1-2 group were 85.44% and 71.66%,and the DDX1 protein levels were decreased by 99.1% and 96.7%,respectively,which significantly decresed compared with that of control group (P<0.01) .Conclusion A glioma cell line with DDX1 gene silencing was successfully constructed,which provided a cellular model to study the role of DDX1 in the biological behavior of glioma.