Effects Of Inhibition Of Cyclooxygenase-2 By ShRNA On Proliferation And Apoptosis Of Hepatocellular Carcinoma Cell HepG₂

Objective To construct the eukaryotic expression plasmid of short hairpin RNA (shRNA) targeted against cyclooxygenase-2 (COX-2) gene (pshRNA-COX-2), and investigate the effects of cyclooxygenase-2 (cox-2) inhibited by shRNA on the proliferation and apoptosis of hepatocellular carcinoma cell HepG2.Methods One pair of shRNA with reverse repeated sequence targeting COX-2 mRNA spaced by 9bp nucleotides were synthesized, the complement double strands were formed by annealing and inserted into plasmid pGenesil-1 to construct the eukaryotic expression plasmid of shRNA targeted against COX-2 gene (pshRNA-COX-2), and transformed into JM109 strain. The recombinant expression plasmid pshRNA-COX-2 was identified by enzyme restriction and sequence analysis. The identified pshRNA-COX-2 plasmid was transfected into hepatocellular carcinoma cell HepG2 with LipofactamineTM2000. The expression of COX-2 mRNA and protein were detected with reverse transcriptional polymerase chain reaction (RT-PCR) and Western blot assay, respectively. The growth of and cell cycle of hepatocellular carcinoma cell HepG2 were detected with cell account and flow cytometry. Cell apoptosis was detected with electron microscope.Results Recombinant expression plasmid pshRNA-COX-2 was successfully constructed. It could inhibit the expression of COX-2 gene. Compared to untransfected group, recombinant expression plasmid pshRNA-COX-2 resulted in reduction of COX-2 mRNA and protein expressions by 69.9% and 50.3% respectively, the growth of HepG2 cells was significantly inhibited, and cells in G0-G1 phase increased from 55.40% to 77.89%, cells in S phase decreased from 31.06% to 16.19%, and appeared apoptosis cells under electron microscope.Conclusion Recombinant expression plasmid pshRNA-COX-2 was constructed. Plasmid pshRNA-COX-2 could significantly inhibit the expression of COX-2 gene, and suppress growth of tumor cells, and induce tumor cell apoptosis, which offered laboratory evidences for tumor gene therapy with RNAi strategy targeted against COX-2 gene.