Effects Of Shrna-VEGF Interference On Cell Growth And VEGF Expression Of Tongue Cancer Tca/DDP Cell Line

Objective:(1)To explore the mRNA and protein expression of vascular endothelial growth factor (VEGF) in human tongue squamous cell carcinoma cell line(Tca8113)and its Cisplatin-induced drug-resistant cell line (Tca/DDP). (2)To transfect short-hairpin RNA(shRNA)eukaryotic expression vector targeting VEGF constructed by the previous experiment into Tca/DDP cells cultured in vitro, as well as to observe the inhibition of VEGF expression and proliferation of Tca/DDP cells.Methods:(1)MTT assay was used to test the sensitivity to different chemotherapeutic medicines of Tca/DDP cells and parental cells;cellular immunohistochemistry was applied to detect multi-drug resistant protein P-gp expression in both parent cells and Tca/DDP cells. (2)Reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot were applied to detect the mRNA and protein of VEGF in Tca8113 cells and Tca/DDP cells cultured in vitro.(3)The VEGF-targeting eukaryotic expression vectors were transfected into Tca/DDP cells by liposome LipofectamineTM 2000. RT-PCR and Western Blot were performed to analyze the inhibition of VEGF expression 48 hours after the transfection mediated by LipofectamineTM 2000. (4) MTT assay was used to explore the effect of interference on Tca/DDP cell growth. Results:(1)MTT assay showed that the tolerance of Cisplatin-induced drug-resisdant cells to different chemotherapeutic drugs had been increased to different extents;expression of P-gp was expressed higher in Cisplatin-resistant cells.(2) The expression of VEGF mRNA and protein in Tca/DDP cells was not significantly different from that in Tca8113 cells.(3)Forty-eight hours after the transfection mediated by LipofectamineTM 2000, RT-PCR and Western Blot showed the mRNA and protein of VEGF in Tca3/DDP cells significantly decreased in the interference group, compared by the non-transfected group, mock control group and scrambled sequence group.(4) MTT assay showed that the growth of Tca/DDP cells of interference group were decreased significantly through inhibiting VEGF expression, compared with that of the non-transfected group, mock control group and scrambled sequence groups 24 hours,48 hours and 72 hours after transfection.Conclusion:(1)Suggesting that Tca/DDP cells have the biological properties of multi-drug resistance.(2) VEGF expression exsisted and had no significant difference in Tca/DDP and Tca8113 cells. (3)The shRNA targeting VEGF, constructed with pGenesil1.1 vector, can inhibit the mRNA and protein expression of VEGF in Cisplatin-induced drug-resistant tongue cancer cells. (4)Targeting of VEGF can decrease the growth of Tca/DDP cells suggesting that targeting inhibition of VEGF gene may lay some theoretical foundation for the research into treatment of cisplatin-resistant tongue squamous cell carcinoma.