shRNA干扰肝癌细胞Kir6.2基因表达载体的构建及鉴定  

Construction and identification of vector against Kir6.2 gene expression in hepatoma cells by short interfering RNAs

作　　者：苏晓通[1] 梁平[1] 李靖[1] 丁生才[1] 杨彤翰[1] 

机构地区：[1]第三军医大学新桥医院肝胆外科,重庆400037

出　　处：《西南国防医药》2008年第3期340-343,共4页Medical Journal of National Defending Forces in Southwest China

摘　　要：目的:构建shRNA体内表达载体pGenesil-3-siRNA,筛选出抑制Kir6.2基因表达的有效靶序列。方法:以Kir6.2基因为目的基因,以产生shRNA质粒载体pGenesil-3为表达模板,细胞内转录合成2条siRNA,脂质体转染法将shRNA质粒载体pGenesil-3共转染SK-Hep1细胞,将细胞分为SK组(未转染)、HK组(转染阴性对照质粒)、K1组(转染重组质粒K1)和K2(转染重组质粒K2)组。经G418筛选出单克隆进行培养,用RT-PCR和Western Blotting分别检测Kir6.2 mRNA和蛋白的表达,筛选出抑制Kir6.2表达的有效siRNA靶序列。结果:SK、HK、K1和K2组Kir6.2 mRNA表达的相对量分别为0.936±0.042、0.848±0.058、0.225±0.0360、0.171±0.029,K1、K2组Kir6.2mRNA的表达与HK、SK组比较均明显降低(P<0.05)。SK、HK、K1和K2组的Kir6.2蛋白表达的量分别为0.925±0.045、0.901±0.067、0.269±0.024、0.318±0.031,K1、K2组Kir6.2蛋白的量与HK、SK组比较均明显降低(P<0.05)。结论:shRNA抑制了肝癌细胞Kir6.2基因的表达。Objective.To construct the expression vector pSlience - 3 - siRNA that can express the short hairpin RNAs （shRNA） against Kir6. 2 gene and to screen the effective target sequence of shRNA_ Methods： Two shRNA against Kir6.2 gene were transcript synthesized intracellularly by expressed templates of plasmid vector pSilence - 3, and the target sequence of Kir6. 2 gene was inserted into the upstream of the reporter gene in order to construct the recombinant plasmid vector pSlience - 3. The plasmids containing 2 different sequences of human Kir6. 2 mRNA coding region were constructed and transfected into Sk- Hepl cells with Lipofectamine 2000 methods, respectively. The cells were divided into 4 groups： SK （normal）, HK （transfected the recombinant plasmid HK）, K1 （transfected the recombinant plasmid K1） and K2 （transfected the recombinant plasmid K2） group. The selected single clone was cultured after screening by G418. The expression of Kir6. 2 gene was detected by semiquantitative reverse transcription - polymerase chain reaction （RT - PCR） and Western blotting. The effective siRNA against the expression of Kir6. 2 was screened out. Results： The relative quantity of the Kir6. 2 gene expression of SK, HK, K1, K2 detected by RT - PCR was 0. 936±0. 042, 0. 848±0. 058,0. 225 ±0. 036, 0. 171±0. 029, respectively. Compared with SK and HK groups, the expression of Kir6. 2 mRNA in K1 and K2 groups decreased significantly （P〈0. 05）. The relative quantity of the Kir6. 2 protein of SK, HK, K1, and K2 was 0. 925±0. 045, 0. 901±0. 067, 0.269±0.024, 0. 318±0. 031, respectively. The quantity of Kir6. 2 protein in Kland K2 groups reduced more than in SK and HK groups（P〈0. 05）. Conclusion. The shRNA can inhibit the expression of Kir6. 2 gene in hepatoma cells.

关 键 词：RNA干扰 肝细胞癌 RT-PCR WESTERN BLOTTING 

分 类 号：R735.8[医药卫生—肿瘤]