DDX46基因慢病毒载体的构建及其在人膀胱癌细胞中的表达

Construction of DDX46 lentiviral vector and its expression identification in human bladder cancer cells

目的构建DDX46基因低表达慢病毒载体,并检测其在人膀胱癌细胞中的表达效果,为DDX46基因在人膀胱癌细胞中的研究奠定基础。方法应用实时荧光定量检测5637细胞和T24细胞中DDX46 m RNA表达水平,以寻找合适的细胞系进行实验。分别以DDX46基因序列和普适型阴性序列为模板,设计合成靶点序列,并合成寡核苷酸双链,定向克隆到慢病毒质粒GV115,合成重组质粒sh DDX46和sh RNA,分别称之为实验组和对照组。将不同重组质粒与pHelper 1.0和pHelper 2.0分别转染293T细胞以包装成慢病毒颗粒后感染5637细胞和T24细胞。应用实时荧光PCR定量检测重组慢病毒感染5637细胞和T24细胞后DDX46 m RNA表达水平,判断其干扰效率。结果 5637细胞和T24细胞均高丰度表达DDX46m RNA,其结果(用ΔCt值表示)分别为(6.53±0.08)和(8.48±0.11);重组慢病毒感染膀胱癌细胞后,在5637细胞中,实验组DDX46 m RNA的表达水平(用ΔCt值表示)为(0.32±0.01),低于对照组(1.00±0.10),差异有统计学意义(P<0.05),其敲减效率为67.70%;在T24细胞中,实验组DDX46 m RNA的表达水平(用ΔCt值表示)为(0.11±0.01),低于对照组(1.00±0.03),差异有统计学意义(P<0.05),其敲减效率为89.00%。结论成功构建DDX46基因低表达慢病毒载体,为研究DDX46基因在膀胱癌细胞中的作用奠定了基础。

Objective To construct lentiviral vector of low expression of DDX46 gene and detect its expression in human bladder cancer cells to explore the effect of DDX46 gene on bladder cancer cells. Methods The expression levels of DDX46 m RNA in 5637 cells and T24 cells were detected by real-time fluorescence to find suitable cell lines to go on. DDX46 gene and universal negative sequencewere used as template to design target sequence to synthesize oligonucleotide duplexes and be cloned into plasmid GV115,then the recombinant plasmids sh DDX46 and sh RNA were synthesized,and all of them combined with pHelper 1 and pHelper 2 were transfected into 293 T cells to package lentivirus particles to infect 5637 cells and T24 cells. The DDX46 m RNA expression levels of 5637 cells and T24 cells transfected by recombinant plasnids were detected by real-time fluorescence quantitative PCR to judge the interference efficiency. Results A total of 5637 cells and T24 cells expressed high abundance of DDX46 m RNA,and their results(expressed by delta Ct values) were(6. 53 + 0. 08) and(8. 48 + 0. 11). After recombinant lentiviral infection trasfecting bladder cancer,the expression level(represented by a ΔCt value)(0. 32 ±0. 01) of DDX46 m RNA in the experimental group in 5637 cells was lower than that in the control group(1. 00 ± 0. 10) in 5637 cells,the difference was statistically significant(P < 0. 05),and the knockdown rate was 67. 70%; the expression level(represented by a Ct value)(0. 11 ± 0. 01) of DDX46 m RNA in the experimental group in T24 cells was lower than that in the control group(1. 00 ± 0. 03) in T24 cells,the difference was statistically significant(P < 0. 05),and the knockdown rate was 89. 00%. Conclusion The lentiviral vector of low expression of DDX46 gene issuccessfully constructed to lay the foundation for studying the role of DDX46 gene in bladder cancer.