Objective: To investigate the inhibitory effect of plasmid-based survivin-specific short hairpin RNA and GRIM-19 on the growth of Hep-2 laryngeal cancer cells.
Methods: The plasmid expressing survivin-specific short hairpin RNA (shRNA) and GRIM-19 (p-siRNA survivin/GRIM-19) was prepared and transfected into Hep-2 cells with Lipofectamine 2000. The mRNA and protein expression of surviving and GRIM-19 were measured with RT-PCR and western blot assay, respectively. MTT assay was employed to detect the proliferation of Hep-2 cells, and flow cytometry and AO/EB assay were done to determine the apoptosis of Hep-2 cells.
Results: In the p-siRNA survivin/GRIM-19, the mRNA and protein expression of survivin was markedly reduced by 54.4% and 42.2%, and the reduction in protein expression of surviving was more obvious than that in the p-siRNA survivin group (37%) (P<0.05). The protein expression of GRIM-19 was markedly enhanced when compared with the control group (P<0.01). MTT assay revealed the proliferation of Hep-2 cells undergoing transfection with p-siRNA survivin/GRIM-19 was markedly inhibited, and the inhibition rate was as high as 79%, which was higher than that in the psi-survivin group (45%) and p-GRIM-19 group (35%). AO/EB assay and flow cytometry indicated that the apoptotic cells in the p-siRNA survivin/GRIM-19 group were dramatically increased as compared to the psi-survivin group and p-GRIM-19 group.
Conclusion: The p-siRNA survivin/GRIM-19 has marked decrease in survivin expression and dramatic increase in GRIM-19 expression. Moreover, silencing of survivin and over-expression of GRIM-19 can significantly inhibit the growth and induce the apoptosis of Hep-2 in vitro and in vivo.
Keywords: Co-expression plasmid; gene silencing; laryngeal cancer; survivin.