Inhibition Of Expression Of VEGF In MCF-7 Cells By SiRNAs

ObjectiveTo specifically inhibit the expression of VEGF in human breast carcinoma cell line MCF-7 with RNAi technology.MethodsTwo siRNAs targeting human VEGF and one scrambled siRNA (used for a positive control) were designed on line. The three kinds of siRNAs were obtained by in vitro transcription and transfected into cells with lipofectin.According to the final concentration of siRNAs, the MCF-7 cells were divided into 7 groups:(1)siRNA₁ or siRNA₂ 100nmol/L group(2)300nmol/L group(3)600nmol/L group (4)900nmol/L group(5)the control scramble siRNA group 300nmol/L(6)lipofectin group: no siRNAs (7_untreaded group: no siRNAs and no lipofectin. We observed and evaluated the survival of the transfected cells with MTT assay, the VEGFmRNA level in the transfected cells with RT-PCR as well as the secretion of VEGF protein in the supernatant with ELISA. In RT-PCR and ELISA we only use one siRNAs' concentration: 300nmol/L. The statistics of variables we have achieved was treated with SPSS 12.0.ResultsThe small interfering RNA (siRNA) targeting human VEGF almost completely inhibited the secretion of VEGF in MCF-7, whereas the control scramble siRNA showed no effects.The OD value of MTT reduction assay showed that siRNA₁ and siRNA₂ markedly inhibited the growth of MCF-7 cells separately while the control scramble siRNA showed no effects to MCF-7 cells. Comparing with the untransfected group, the siRNA₁ group and the siRNA₂ group had statistical significance separately: (1)-(3)P<0.01 (4)P<0.05;the control scramble siRNA P>0.05. RT-PCR data showed that VEGFmRNA level was markedly downregulated in MCF-7 cells transfected with siRNA₁ and siRNA₂ for 24 hours, while the control scramble siRNA showed no effects to MCF-7 cells .The ELISA data showed the secretion of VEGF protein in the supernatant of the MCF-7 cells transfected with siRNA₁ and siRNA₂ for 24 hours were significantly lower...