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

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-Hep1 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(P0.05).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 K1and K2 groups reduced more than in SK and HK groups(P0.05).Conclusion:The shRNA can inhibit the expression of Kir6.2 gene in hepatoma cells.