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

Objective:We develop a novel Vasular Endothelial Growth Factor-C (VEGF-C) blockade method using RNA interference(RNAi) in a human gastric cancer cell line,SGC7901 in vitro. Its effects and effective small interfering RNA (siRNA) are examined. We establish a SGC7901 s.c. xenograft model of BALB/c nude mouse, and Study on whether inhibition of VEGF-C using injection of siRNA suspension intratumaorlly might lead to inhibition of tumor growth and tumor lymphangio- genesis in gastic cancer in vivo.Methods:In Vitro: We designed and chemically synthesised VEGF-CsiRNA. And siRNA was purified by native gel electrophoresis(PAGE); SGC7901 cells were transfected using liposome-mediated siRNA. After siRNA was transfected, we observed the morphous of SGC7901 cells and measured cell proliferation by Microculture Tetrazolium Thiazolylblue(MTT); SGC7901 cells were tansfected using 6-Fam dioxyfluoran-labeled siRNA, and cells were observed through fluorescence microscope to calculate efficiency of transfection. The expression level of VEGF-C mRNA was mesured by semi-quantitative RT-PCR in each group of various siRNAs transfected; Variation of VEGF-CmRNA and protein level accompanied by variation of time were mesured by semi-quantitative RT-PCR and Western blot. In vivo: 24 SGC7901 s.c. xenograft models of BALB/c nude mouse were estabished first of all. They were divided into 4 groups in random:group of injection of siRNA-liposome-suspension; group of liposome only; group of siRNA only;groups of PBS injection. VEGF-CsiRNA suspension were injected intratumorally when the diameter of tumor reached 0.5cm. It performed every 6 day per time repeatly,and 3 times allover.Sequentially the accumulation of tumor volume of each experiment group was obeserved and the size was recorded. The expression of VEGF-C in tumor tissue was measured by Immunohistochemical (IHC).And the status of Angiogenesis and Lymphangiogenesis were examined by IHC dyeing using human anti-CD31 monoclonal antibody and anti-LYVE-1 monoclonal antibody.Results:In Vitro:After transfected with liposome-mediated VEGF-CsiRNA, decreased growth of SGC7901 cells emerged, but MTT result show that there is on obviously defference among group of non-transfected, group of liposome using only and group of VEGF-CsiRNA-transfected(P<0.05). SGC7901 cells was tansfected by 6-Fam dioxyfluoran-labeled siRNA and GAPDHsiRNA eachly. And It shows that working concertration is optiming at 100mmol/L with its efficiency transfection: 37.03%±3.0%,and the mRNA level of endogenous gene GAPDH was down-regulated to 65.5%±3.3% compared with group of non-transfected.The effective VEGF-CsiRNA detected by semi-quantitative RT-PCR down-regulated the level of VEGF-CmRNA to 52.5%±2.6% compare with control group, and the climax of knockdown effect that reach to 43.5% emerge at 48 hours after siRNA transfection; Also,the level of VEGF-C protein is down-reglated to 32.7%±1.1% compared with control group,and the knockdown effect that reach to 18.9% emerge at 72hour after siRNA transfection.In Vivo: Tumor growth in intervention group is obviously inhibited by injection of VEGF-CsiRNA suspension intratumorally compared with control group (P<0.01).There is on obvious difference in group of using liposome or no liposome(P>0.05).The expression of VEGF-C in intervention group using anti-VEGF-C monoclonal antibody immunohistochemistry dyeing is downregulated to 68.3%(P<0.01)compared by control group. Lymphangiogenesis in intervention group is also reduce through detection of anti-LYVE-1 monoclonal antibody immunohistochemistry Value of LVD is 3.2±1.3 in intervention group,compared 9.8±2.7 in control group, and reduce to 32.7%(P<0.01). But there is no obviously variance in MVD value between intervention group and control group(P>0.05). Conclusion : The screened VEGF-CsiRNA can dramatically down-regulate the expression of VEGF-CmRNA and protein. In vitro inhibition the expression of VEGF-C lead to decreaced growth of SGC7901 cells , but not to induce cell apoptosis. In vivo, The VEGF-CsiRNA dramatically downregulated expression of VEGF-C in tumor tissue.And It lead to inhibition of tumor growth and tumor Lymphangiogeneis ,but on effect on angiogenesis.