Effect Of Suppressing COX-2 Expression By ShRNA On Adhesion And Invasion Of Human Hepatocellular Carcinoma Cell

Part I Construction and identification of shRNA expression vector targeted to COX-2 geneObjective To construct the eukaryotic expression plasmid of shRNA specific to human COX-2 gene and observe its inhibitory effect on the expression of COX-2 in human hepatocellular carcinoma cells.Methods Two shRNA template sequence of COX-2 were synthesized and inserted into the vector pGenesil-1 to generate the plasmids named WBH1 and WBH2. The plasmid HK was used as control group. The recombinant plasmids were transfected into human hepatocellular carcinoma cell lines HepG2 and BEL7402.The transfection efficiency was detected. The expression of COX-2 was assayed by RT-PCR and Western blot after HepG2 and BEL7402 cells were transfected with liposomes for 24, 48, 72 and 96 hours. Results The shRNA express vectors had been successfully constructed by restriction endonuclease analysis and DNA sequencing. The transfection rates in HepG2 and BEL7402 cells after 48 hours were about 60% and 54% respectively.The inhibition efficiencies of plasmid WBH1 in HepG2 and BEL7402 by RT-PCR were 18.5%, 88.6%, 52.8%, 42.4%(P<0.01) and 9%, 45.1%, 70.1%, 56.3% (P<0.01), compared with those of the control at 24, 48, 72 and 96 hours.The inhibition efficiencies of plasmid WBH1 in HepG2 and BEL7402 cells by Western blot were 10.2%, 80.5%, 45.3%, 39.0% (P<0.01) and 8.3%, 40.2%, 66.4%,35.6%at 24, 48, 72 and 96 hours(P<0.01). The plasmid WBH2 had no effect on COX-2 expression.Conclusion COX-2 expression in human hepatocellular carcinoma cells can be inhibited significantly by construction of eukaryotic expression vector expressing the shRNA. The inhibitory effect is highly related with selection of target sequence.Part II: ShRNA targeted to COX-2 influence the adhesion and invasion of human hepatocellular carcinoma cell line HepG2Objective To study the effect of COX-2 shRNA on the cell adhension and invasion in human hepatocellular carcinoma cell line HepG2 in vitro.Methods The plasmids WBH1 and HK were transfected into HepG2 cells .The untransfected cells were used as control group. At 48h after transfection, the ability of adhesion on extracellular matrix matrigel was detected using MTT assay. Invasive ability was measured by the number of cells cutting through artificial membrane. Results The adhesive rate of WBH1-transfected group obviously reduced by 47.4% compared with control group [(6.0±0.4)%vs(11.4±0.2)%, P<0.01]. There was marked difference between WBH1-transfected group and control group on the cell number that infiltrated the membrane (8.25±1.50vs22.75±1.70, P<0.01) and the inhibition rate was 63.7%. But there was no difference between the above-mentioned indexes of HK-transfected group and those of control groupConclusion ShRNA targeted to COX-2 can inhibit the adhesiveness and invasiveness of human hepatocellular carcinoma cell line HepG2.