RNAi-mediated Gene Silencing Of Vascular Endothelial Growth Factor Inhibits Proliferation And Reduces Angiogenesis In Colon Cancer Cells

Objective: There are many reasons for malignant proliferation of colon carcinoma. Vascular endothelial growth factor (VEGF) has been implicated as a critical molecular signal in tumour development, by promoting angiogenesis and possibly by exerting autocrine functions in tumour cells. Some studies showed that VEGF was over expressed in colon cancer. Therefore, blocking the expression of vascular endothelial growth factor might be an effective therapeutic strategy for colon cancer. The human VEGF gene has been assigned to chromosome 6p12. It is formed of eight exons, separated by seven introns, and its coding region spans approximately 14 kb. Five VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene: VEGF121, VEGF145, VEGF148, VEGF165, VEGF183, VEGF189 and VEGF206. VEGF165 are the main proteins synthesized and secreted by colon cancer cells. In recent years, the RNA interference technique has become a new and hot research field for gene therapy because it can block the target gene expression efficiently and specifically. This new approach has been successfully applied to give specific inhibition of the expression of exogenous and endogenous genes in mammalian cells. To test the potential for this type of gene therapy, we constructing a eukaryotic expression plasmid containing short interfering RNA (siRNA). Evaluate the effects of this vector on the proliferation and angiogenesis of colon cancer in vitro and in vivo.Methods: The Pavu6+27-VEGF165 siRNA expression vector was constructed by gene recombination and transfected into colon cancer cell line HCT116 by liposome. The positive transfected cell clones were screened with G418. Expression of VEGF mRNA and protein in HCT116 cells after gene transfer was detected by semi-quantitative reverse transcription–polymerase chain reaction and Western blot assay, respectively. Proliferation of pAVU6+27-VEGF165-siRNA-transfected HCT116 cells and control cells was analysed by MTT assay. Flow cytometry (FCM) was used to analyse the distribution of cell cycle of...