Effect of knockdown of VEGF gene expression by shRNA on characteristics of human colorectal cancer cell line

Objective To study the effect of knockdown of VEGF expression by recombinant plasmid Pavu6+27-VEGF siRNA on the characteristics of human colorectal carcinoma cell line HCT116. Methods The subcloned recombinant plasmid shRNA was transfected into human colorectal carcinoma cell line HCT116 with DOTAP method. The positive transfected cell clones were screened with G418. The stable transfection and expression of VEGF mRNA in HCT116 were determined by semi-quantitative RT-PCR. The protein expression of VEGF was examined by Western blotting. Distribution of cell cycle was assessed by flow cytometry. The cell growth suppression was analysed by MTT assay. Results Compared with the negative control cells, multi RT-PCR and Western blotting showed the expression of VEGF mRNA and protein was down-regulated in the transfected cells (P0.05). The subcloned recombinant plasmid expressing shRNA effectively inhibited HCT116 cell growth and proliferation while empty plasmid had no such specific effect. The proliferation index of the cellls of recombinant plasmid transfection was (25.63±4.54)%, while that of the negative control cells was (31.90±2.19)% (P0.05). Conclusion Recombinant plasmid Pavu6+27-VEGF siRNA in cells specially inhibit the expression of VEGF mRNA in HCT116 cells, the expression of VEGF in cell culture fluid and the growth of cell line HCT116.