Effect of shRNA silencing expression of Kir6.2 gene in hepatoma cells on the intracellular calcium concentration

Objective To study shRNA silencing expression of Kir6.2 gene in hepatoma cells and the effect on the intracellular calcium concentration.Methods Plasmid vectors containing negative control sequence HK and 2 kinds of different sequences of K1 and K2 were constructed and transfected into Sk-Hep1 cells by using the Lipofectamine 2000 method,respectively.The experiment was 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).The selected single clone was cultured after screening by G418.The expression of Kir6.2 gene was detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting.The effective shRNA inhibiting the expression of Kir6.2 was screened out.Fluo4-AM incubated the cells in every group.Fluorescent intensity was detected by flow cytometry.Results Compared with SK and HK groups,the expression of Kir6.2 mRNA in K1 and K2 groups significantly decreased(P0.05).The quantity of Kir6.2 protein in K1 and K2 groups reduced more than in SK and HK groups(P0.05).Calcium concentration in K1 and K2 groups was much lower than that in SK group.Calcium concentration in SK,HK groups was remarkably higher than that in K1 and K2 groups.Conclusion The shRNA can inhibit the expression of Kir6.2 gene in hepatocellular carcinoma.Intracellular calcium concentration is dramatically low.