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Cyclooxygenase-2 gene silence induces apoptosis of human hepatocellular carcinoma cells

Keywords: cyclooxygenase 2 , shRNA , carcinoma , hepatocellular , apoptosis , bcl-2-associated X protein

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Objective To specifically silence COX-2 gene expression in cultured human hepatocellular carcinoma cells HepG2 by construction and transfection of COX-2 hairpin RNA (shRNA) expression vectors, and investigate the effects of knocking COX-2 down on cell proliferation and apoptosis. Methods The inserted sequences coding shRNA homologous to COX-2 gene were designed and COX-2 shRNA expression vectors were constructed and transfected into HepG2 cells with Lipofectamine 2000. The inhibitory efficiency of COX-2 shRNA was determined by detection of COX-2 mRNA levels in transfected cells using semi-quantitative RT-PCR. Cell viability was analyzed by cell counting kit-8 (CCK-8) assay. Indexes of apoptotic cells and cell cycle distribution were analyzed by flow cytometry. Western blotting was used to detect the changes of protein levels of COX-2 and Bcl-2 in HepG2 cells. Results Plasmid sequencing indicated the successful construction of two shRNA expression vectors. The expressive levels of COX-2 mRNA in HepG2 cells transfected with two COX-2 shRNA expression vectors decreased to 41.66% and 77.79%, respectively, of that in cells transfected with control scrambled shRNA expression vector. The vector which efficiently suppressed the expression of COX-2 mRNA was transfected into HepG2 cells, and G418 was used to select a population of cells which may stably express the shRNA. The expression of COX-2 mRNA decreased by 75.30% and the cell viability decreased by 29.33% in cells which stably expressed COX-2 shRNA. Flow cytometry analysis showed a higher proportion of apoptotic cells in COX-2 shRNA group than in untransfected and scrambled shRNA groups (17.83% vs4.47% and 4.63%). The proportion of cells in G0/G1 phase was 73.93%, 57.63% and 61.2%, while in S phase was 13.43%, 26.90% and 26.03%, respectively, in COX-2 shRNA group, untransfected and scrambled shRNA groups. Western blotting revealed that the protein levels of COX-2 and Bcl-2 in HepG2 cells which expressed COX-2 shRNA reduced to 31.25% (P < 0.01) and 54.93% (P < 0.01) of scrambled shRNA expression vector respectively. Conclusion Recombinant expression vector pSilencer 2.1-U6-COX-2 shRNA may efficiently silence COX-2 gene expression, inhibit cell proliferation, promote apoptosis, arrest cell cycle and decrease the Bcl-2 protein level in human hepatocellular carcinoma cells HepG2.


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