Livin基因siRNA慢病毒表达载体的构建及其对喉癌细胞HEP-2增殖的影响

Construction of Livin gene siRNA lentiviral vector and its influence on proliferation of HEP-2

目的构建及鉴定Livin基因siRNA慢病毒表达载体并观察其对喉癌细胞HEP-2增殖的影响。方法从基因库获得Livin基因序列,设计3段siRNA序列;经MluⅠ和ClaⅠ双酶切后克隆入慢病毒表达载体p Len OR-THM,构建p Len OR-THMLivin-siRNA重组质粒,将其转化感受态细菌DH5α,对阳性克隆进行筛选后,用EcoRⅠ和XbaⅠ双酶切及测序鉴定正确后用脂质体转染293T细胞包装病毒颗粒,感染293T细胞及HEP-2细胞,用荧光定量PCR检测其对HEP-2增殖的影响。结果慢病毒表达载体p Len OR-THM-Livin-siRNA被成功构建;转染293T细胞组装病毒液有绿色荧光,并主要表达在细胞膜上;浓缩后的病毒液感染293T细胞及HEP-2细胞均有绿色荧光表达。SH2-R组的Livin蛋白相对表达量最低为(12.31±4.43)%;SH2-R组与SH2-F、NC组比较,差异有统计学意义(P<0.05)。结论成功构建了Livin基因siRNA慢病毒载体;HEP-2细胞Livin蛋白表达受到抑制。

Objective To construct and identify lentivirus vector of Livin gene siRNA and investigate its effect on the proliferation of HEP-2 cells.Methods Livin gene sequences were obtained from GeneBank, and three siRNA target sequences were designed. After Mlu Ⅰ and Cla Ⅰ double digestion, the lentivirus expression vector pLenOR-THM was cloned. After siRNA sequences were transformed into the competent bacteria DH5 alpha, the candidate clones were identified by DNA sequencing.Using Eco R Ⅰ and Xba Ⅰ double enzyme digestion and sequencing positive clone, the recombinant plasmid were transfected into 293T cells by liposome 2000.Then the 293T cells and HEP-2 cells were infected with the recombinant plasmid. The effect of Livin-siRNA on the proliferation of HEP-2 was detected by fluorescence quantitative PCR. Western blot was used to analyze the Livin protein expression in SH2-R, SH2-F and NC in laryngeal squamous cell carcinoma.Results The lentiviral expression vector pLenOR-THM-Livin-siRNA was successfully constructed and transfected into 293T cells. Strong green fluorescence was observed in the 293T cells under fluorescent microscope after co-transfection with the plasmids of lentiviral vector in the293T cells and HEP-2 cells, which was mainly expressed on the cell membrane. The 293T cells and HEP-2 cells infected by the concentrated virus solution had green fluorescence expression. The relative expression of Livin protein was the lowest in SH2-R[（12.31±4.43）%],and there was a significant statistical difference between SH2-R and SH2-F, NC（ P 〈0.05）.Conclusion The lentiviral vector of Livin-siRNA has been successfully constructed and identified. The siRNA can reduce the expression of Livin protein in laryngeal squamous cell carcinoma.