The Inhibition Of Small Interference RNA On VEGF Expressed In Glioma Cells U251 And It's Effect On The Growth Of Glioma

[Objective] To explore the effect of vascular endothelial growth factor (VEGF) on glioma cells U251 by inhibiting the expression of VEGF using specific small interference RNAs (siRNAs) selected. To investigate the feasibility of targeting VEGF as a gene therapy for glioma by injecting VEGF-siRNA into subcutaneous U251 cell tumors in nude mice.[Methods] Part one: 3 pairs of siRNAs targeting VEGF were designed and chemically synthesized in vitro and a pair of FAM-siRNA as control. These siRNAs were transfected into U251 cells by using OligofectamineTM reagent. The transfecting efficiency was determined by fluorescent microscopy and flow cytometry. The expression of mRNA for VEGF was detected by RT-PCR in different transfectants treated with different VEGF-siRNAs. The most effective siRNA was selected and transfected into U251 cells at different concentrations. The expression of VEGF mRNA was detected by RT-PCR and the protein expression of VEGF was detected by ELISA. Meanwhile, cell apoptosis rate and the change of cell cycle were analyzed by flow cytometry.Part two: Subcutaneous U251 cell tumors were induced by inoculation of 106 cells. VEGF-siRNAs were injected intratumorally at 2.5ug, 4.0ug and 5.0ug, respectively. The mechanism of down-regulation of VEGF inhibiting the growth of gliomas was studied ,which might act as a foundation as a gene therapy for gliomas.[Results] Part one: The result detected by fluorescent microscopy and flow cytometry showed that the transfection efficiency was over 95%. The expression of VEGF mRNA decreased in different degrees after various sequences of siRNAs transfected into U251 cells by 200nM. The most effective siRNA could cause VEGF inhibition by over 70%.The expression of VEGF mRNA decreased by 16%～73% after the siRNA transfected into U251 cells by different concentrations.The expression of VEGF protein decreased by 62.7%. Cell apoptosis could be induced from 8.14±1.10 in blank control group and 28.93±8.35 in negative control group to 63.06±7.12 in VEGF -siRNA transfected group.Part two: The growth of the glioma in nude mice was inhibited at a higher concentration of VEGF-siRNA. Compared to control group, the inhibiting effeciency of VEGF-siRNA was 37.1% and 57.4% at 4.0ug and 5.0ug, respectively. The angiogenesis count decreased in VEGF-siRNA injected groups at higher concentrations.[Conclusions](1) The siRNA targeting VEGF could inhibit the expression of VEGF in glioma cells U251 and induce the apoptosis of U251 transfectants, which was prominent in vitro.(2) The VEGF- siRNA could significantly inhibit the growth of the glioma and the ability of tumorigenicity in nude mice; the amount of the angiogenesis in the tumor was reduced in VEGF -siRNA injected group.