Inhibitory Effects Of Cyclooxygenase-2 Inhibited By ShRNA On The Growth And Angiogenesis Of Human Liver Cancer Cell Subcutaneous Xenograft Tumors In Nude Mice

Objective: To investigate the inhibitory effects of recombinant interference plasmid pshRNA-COX-2 on the growth and angiogenesis of human liver cancer cell Hep3B subcutaneous xenograft tumors in nude mice and the promotive effect on the tumor cells apoptosis.Methods: The constructed recombinant interference plasmid pshRNA-COX-2 and negative control plasmid pshRNA-HK were transfected into Hep3B cells respectively after they were identified by sequence analysis, then monoclonal cell lines were selected by G418 pressure. The expressions of COX-2 mRNA and protein were measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively, the VEGF mRNA expression was detected by RT-PCR. The selected monoclonal Hep3B cells and untransfected Hep3B cells were transplanted into the subcutaneous tissue of nude mice respectively, and xenograft tumor volumes were measured every three days and the growth curves of tumors were drawn. The mice were killed after four weeks. The expressions of COX-2 mRNA and protein in tumor tissue were analyzed with RT-PCR and Western blot respectively, the expression of COX-2 protein and microvessel density (MVD) in xenograft tumors were observed by immunohistochemistry, the tumor cells apoptosis was detected by electron microscope observation and TdT-mediated dUTP nick end labeling in situ assay (TUNEL).Results: Compared to the Hep3B cells without transfected by plasmid pshRNA-COX-2, the inhibition rates of COX-2 mRNA and protein expressions were 65.3% and 52.8% respectively in the pshRNA-COX-2 group (P<0.05), the inhibition rate of VEGF mRNA expression was 56.5% in the pshRNA-COX-2 group (P<0.05). Compared to the Hep3B cells transfected by the negative control plasmid pshRNA-HK, the inhibition rates of COX-2 mRNA and protein expressions were 54.6% and 43.5% respectively in the pshRNA-COX-2 group (P<0.05), the inhibition rate of VEGF mRNA expression was 44.6% in the pshRNA-COX-2 group (P<0.05). The ultimate tumor volume in the pshRNA-COX-2 group was apparently smaller than that of the pshRNA-HK group and the untreated group (P<0.01). Compared to the untreated group, the inhibition rates of COX-2 mRNA and protein expressions in tumor tissue were 48.5% and 61.4% respectively in the pshRNA-COX-2 group. Compared to the pshRNA-HK group, the inhibition rates of COX-2 mRNA and protein expressions in tumor tissue were 41.8% and 55.7% respectively in the pshRNA-COX-2 group. The COX-2 score and MVD in the pshRNA-COX-2 group were significantly lower than that of the pshRNA-HK group and the untreated group (P<0.01). The apoptosis cells increased in the pshRNA-COX-2 group observed with electron microscopy, and the results of TUNEL revealed that the apoptosis index (AI) of tumor cells in the pshRNA-COX-2 group tumors was much higher than those of the pshRNA-HK group and untreated group (P<0.01).Conclusions: The plasmid pshRNA-COX-2 can significantly inhibit the growth and angiogenesis of human liver cancer cell Hep3B subcutaneous xenografts in nude mice through target inhibiting COX-2 expression, meanwhile can significantly promote the tumor cells apoptosis.