[Effects of COX-2 gene silencing by shRNA on biological characteristics of human hepatocellular carcinoma cell line HepG2].

BACKGROUND & OBJECTIVE: Cyclooxygenase-2 (COX-2) protein is highly expressed in hepatocellular carcinoma (HCC). It may be involved in tumorigenesis and development of HCC. This study was to explore the effects of COX-2 short hairpin RNA (shRNA) on COX-2 expression in HCC cell line HepG2 and on adhesiveness and invasiveness of HepG2 cells in vitro.

METHODS: Plasmids WBH1 and WBH2 containing 2 different sequences of human COX-2 mRNA coding region were constructed, and transfected into HepG2 cells, respectively. The expression of COX-2 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot at 24, 48, 72 and 96 h after transfection. At 48 h after transfection, the adhesiveness of HepG2 cells to extracellular matrix matrigel was detected using MTT assay. Invasiveness was measured by Transwell experiment.

RESULTS: The transfection rate in HepG2 cells was about 60%. The inhibition rates of COX-2 mRNA expression in HepG2 cells were 18.5%, 88.6%, 52.8%, and 42.4% at 24, 48, 72 and 96 h after transfection of plasmid WBH1 (P<0.01); the inhibition rates of COX-2 protein expression were 10.2%, 80.5%, 45.3%, and 39.0%, respectively (P<0.01). Plasmid WBH2 had no significant inhibitory effect on COX-2 expression in HepG2 cells (P>0.05). The adhesion rate of WBH1-transfected cells was obviously reduced by 47.4% of control cells [(6.0+/-0.4)% vs. (11.4+/-0.2)%, P<0.01]. The cell number that infiltrated Transwell membrane in WBH1-transfected group was significantly reduced by 63.7% of control group (8.25+/-1.50 vs. 22.75+/-1.70, P<0.01). WBH2 had no obvious effects on adhesiveness and invasiveness of HepG2 cells.

CONCLUSION: COX-2 shRNA can inhibit the adhesiveness and invasiveness of HepG2 cells through intervening the expression of COX-2.