Downregulating The Expression Of VEGF In HT-29 Colon Carcinoma Cells By Specific Small Interfering RNAs

ObjectiveTo investigate the specific inhibition of the expression of key gene in the angiogenesis of human carcinoma cell line cultured in vitro with RNAi technology and prove the possibility of RNAi in cancer therapy.MethodsSpecific small interfering RNA was introduced with VEGFmRNA as a target in cultured human colon carcinoma cell line HT-29. Two siRNAs and one scrambled siRNA (used for a negative 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 HT-29 cells were divided into 7 groups:(1) siRNA₁ or siRNA₂ 100nmol/L group(2)siRNA₁ or siRNA₂ 300nmol/L group(3) siRNA₁ or siRNA₂ 600nmol/L group(4) siRNA₁ or siRNA₂ 900nmol/L group(5)scrambled siRNA control group 300nmol/L(6)lipofectin control group: no siRNAs ?untreated control 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 markedly inhibited the secretion of VEGF in HT-29 cells, whereas the control scramble siRNA showed no effects.The OD value of MTT reduction assay showed that siRNAi and siRNA 2 evidently inhibited the growth of HT-29 cells separately while the control scrambled siRNA showed no effects to HT-29 cells. Compared with the scrambled siRNA group, the siRNA₁ group and the siRNA₂ group had statistical significance , respectively, (1)-(3) P<0.01 (4)P<0.05;but the untransfected group siRNA had no statistical significance with...