Down-Regulation Of Vascular Endothelial Growth Factor-C Using Adenovirus-mediated Small Interfering RNA Inhibits Tumor Lymphangiogenesis Of Colon Cancer

Objective:First of all,to construct recombinant adenovirus containing small interfering RNA(siRNA)targeting human VEGF-C mRNA.Then,in vitro,to observe the inhibitory effects of expression of VEGF-C after transfecting LOVO cells;In vivo,to study the inhibitory effects of adenovirus-mediated VEGF-C siRNA on growth and lymphangiogenesis of human colon cancer in BABL/c nude mice model.Methods:In vitro:The specific siRNA sequence targeting human VEGF-C mRNA was selected.The homologous double-strand DNA was designed and synthesized.After such DNA was inserted into pDC316-EGFP-U6 by BamHI and HindⅢ, pDC316-VEGF-C siRNA-EGFP-U6 was obtained,then it was co-transfected into 293 cells with the bone plasmid pBHGF35.After generated by homologous recombination, Ad5F35-VEGF-C siRNA-EGFP-U6 was obtained,and it was packaged and amplified in 293 cells.Ad5F35-VEGF-C siRNA-EGFP-U6 was transfected into LOVO cells.Tumor cells morphologic change was examined and transfection efficiency was calculated with fluorescence microscope.The level of VEGF-C mRNA in cells was tested by Real Time PCR and the concentration of VECF-C protein in the cells was determined by Wetsern-blot;In vivo:The human colon cancer model was established in nude mice by hypo-injection LOVO cells.They were divided randomly into four groups(n=6):adenovirus,virus without target gene,single siRNA and PBS control group. Injecting intervention intra-tumorly in each groups,calculating the tumors volume and drawing the growth curve,calculating micro-vascular density(MVD)and micro-lymphatic density(LVD)by staining respectively of the lymphatic and vascular with anti-LYVE-1 monoclonal antibody and anti-CD34 monoclonal antibody were completed.Results:In vitro:The recombinant adenovirus Ad5F35-VEGF-C siRNA-EGFP-U6 was successfully constructed with the titer of 7.9×10~9 IU/ml by purify.The efficiency of transfection up to the peak when the MOI equal to 100.The adenovirus transfection efficiency to LOVO cells(green fluorescent cells / total cells)was 92%±4.6%.The VEGF-C mRNA expression was decreased to 30.7%±1.2%(P＜0.05)of the control group 48 hours later and the VEGF-C protein concentration was decreased to 47.8%±2.2%(P＜0.05)of the control group 72 hours later;In vivo,adenovirus group tumors sizes were smaller than other groups.The LVD were individually 8.47±2.1 and 17.35±4.7(p＜0.05)in adenovirus group and the PBS control group.The MVD were respectively 22.65±6.04 and 23.19±7.63(p＞0.05)in adenovirus group and the PBS control group.Conclusions:VEGF-C RNAi mediated by adenovirus is capable of knocking down VEGF-C expression remarkablely in human colon cancer cells.The adenovirus-mediated VEGF-C-siRNA can significantly inhibit the growth and lymphangiogenesis of human colon cancer in nude mice model,but have no effect on microangium vessels growth.