Using Sirna To Inhibit Vegf-c Anti-breast Cancer Angiogenesis Research

Background The vascular endothelial growth factor-C(VEGF-C) promotes the formation of lymphatic vessel of the tumor through its control over proliferation and migration of the new lymphatic vessel endothelial cells of stromal tumor.Utilizing chemically modified small interference RNA(siRNA),the research inhibited expression of VEGF-C in vitro and in vivo to investigate the feasibility and specificity of breast cancer gene therapy.Method siRNA targeting VEGF-C gene was designed.In vitro,ds-siRNA was transfected into MCF-7 cells using cationic liposome jetPEI^TM 2000 as transfecing agent.Set up the Blank control group(Medium of no blood-serum and no antibiotic),Lipofectamine control group(only jetPEI^TM 2000),siRNA concentration gradient group(50,100,200 nmol/L) and siRNA SCR group(100 nmol/L).The proliferation of MCF-7 cells was detected by MTT colorimetry,and the apoptosis was measured by Hoechst33258 staining.Analyze VEGF-C and p53 gene mRNA level by RT-PCR and detect the expression of VEGF-C and p53 gene protein by Western blotting.In vivo,subcutaneously plant MCF-7 cells into the female nude mice to prepare animal model,divide the nude mice with positive tumor into three groups at random:VEGF-C siRNA treatment group;liposome group and control group,with 5 mice each group.VEGF-C siRNA treatment group were injected the mixture of VEGF-C siRNA 1 mg/kg and jetPEI^TM in the local of the tumor,liposome group were injected jetPEI^TM 2000(concentration is alike with the above-mentioned treatment group) and PBS in the local of the tumor,control group only were injected PBS.Inject three times each day,and inject continuously for 8 times.Kill the mice by pulled their necks after 22 days.Analyze VEGF-C mRNA level by semi-quantitative RT-PCR and detect the expression of VEGF-C protein by Western blotting.Result In vitro,the experimental results showed:VEGF-C siRNA effectively inhibited the proliferation of MCF-7 cells.The inhibitory rate was 26.12%,50.01%,52.21%.VEGF-C siRNA got the best inhibiting effect when its final concentration is 100nmol/L(50.01%).Seventy-two hours after siRNA transfection,apoptosis bodies were observed in stained cells by Hoechst33258 under a fluorescent microscope.RT-PCR experimental results showed:compared with the control group,the VEGF-C and p53 gene mRNA expression of VEGF-C siRNA treatment group decreases significantly(P＜0.01).Western blotting experimental results showed:compared with control group, there is a significant decrease of the VEGF-C protein expression in the VEGF-C siRNA treatment group(P＜0.01).In contrast,there were no obvious changes in Blank control group,Lipofectamine control group and siRNA SCR group.In vivo,the experimental results showed:the growth of tumor in treated group by siRNA has been inhibited evidently,the expression levels of mRNA and protein of VEGF-C has decreased significantly(P＜0.05),no significant changes in control group.Conclusion RNAi mediated by chemically modified siRNA markedly decrease VEGF-C gene expression in vitro and in vivo,and inhibited MCF-7 cellular proliferation.It might be a potential new method to tumor gene therapy for siRNA to inhibit expression of VEGF-C.And this study show that p53 gene related with VEGF-C gene expression in breast cancer,p53 gene possible participate the regulation of VEGF-C gene,accordingly participate the vascular growth regulation for breast cancer.