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Sirna-Mediated Gene Silencing In Colorectal Cancer: A Novel Anti-Angiogenic Targeted Therapy

Abby Mulkeen MD, Terry Silva BA, John Schmidtz PhD, Edward Uchio MD, Edward Chu MD, Charles Cha MD
Department of Surgery, Yale University

RNA interference (RNAi) allows for the specific targeting and silencing of genes, mediated by small interfering RNA (siRNA). VEGF is one of the most potent pro-angiogenic factors for tumor growth and is expressed in virtually all solid tumors. Furthermore, recent clinical trials have demonstrated efficacy of anti-VEGF therapy in colon cancer. We therefore investigated the effect of siRNA mediated silencing of VEGF in human colorectal cancer cells.

Methods: siRNAs, designed in silico using a bioinformatics approach, were synthesized as double stranded RNA and screened for VEGF gene silencing efficacy in human colon cancer cells. VEGF expression was quantified by sandwich ELISA analysis and confirmed by Northern blot analysis. Tumor proliferation was measured by WST proliferation assay. Apoptosis was assessed by Caspase-3 and PARP assays.

Results: A series of VEGF specific siRNAs were synthesized targeting the coding region of VEGF and four showed evidence of significant gene silencing in a variety of human colon cancer cell lines, particularly the RKO line. Greater than 95% knockdown was seen on ELISA assay with some sequences, and gene silencing occurred in a dose-dependent manner. A significant effect on tumor proliferation was seen on WST assay with treatment with VEGF targeted siRNA compared to a scrambled control siRNA sequence, as seen in the figure (p=.033), and 0.1 nM siRNA was sufficient to show some effect. It does not appear as if this decrease in proliferation is due to apoptosis. We are currently testing the efficacy of these siRNAs in inhibiting tumor growth in a mouse xenograph tumor model.

Conclusion: SiRNA targeted therapy is effective in silencing VEGF gene expression leading to decreased tumor proliferation in human colorectal cancer cells. This approach represents a potentially effective therapeutic approach to the treatment of a broad range of malignancy.

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