The Study Of VEGF-C-siRNA Inhibiting Proliferation And Lymphatic Metastasis In Human Breast Cancer

At the time of diagnosis,the majority of breast cancer patients have developed lymph node metastasis.Dissemination to regional lymph nodes via afferent lymphatics is the first step in breast cancer metastasis and an important prognostic indicator.Two members of the vascular endothelial growth factor(VEGF) family,VEGF-C and VEGF-D,have been associated with tumor lymphangiogenesis and metastasis.The VEGF-C protein belongs to the platelet-derived growth factor family and is a known ligand for VEGFR-3(fms-like tyrosine kinase,flt-4) located at endothelium of lymphatic vessel.VEGF-C-dependent activation of VEGFR-3 stimulates growth of lymph endothelial cells and lymphatics.The levels of VEGF-C in primary tumors have significantly correlated with lymph node metastasis in a variety of cancers,including thyroid,prostate,gastric, colorectal,lung,and breast cancer.Overexpression of VEGF-C in breast cancer cells can potentially increase intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes.RNAi(RNA interference) is a post-transcriptional gene silencing induced by introduction of double-stranded RNA(dsRNA).RNAi can down-regulate gene expression in cell,which not only be a powerful tool in VEGF-C gene functional research,but also provides a new technique for gene therapy.In the present study,we examined the expression of VEGF-C in human breast cancer tissues and cell lines.We found that the expression VEGF-C was much higher in breast cancer than in normal tissues,especially in breast cancer with lymphatic metastasis.The expression of VEGF-C decreased after new-adjuvant-chemotherapy.Among human breast cancer cell lines,MDA-MB-435 expresses higher level of VEGF-C and is liable to lymphatic metastasis.So we chose this cell line for next experiments.Three small fragments of siRNi and one negative control were synthesized and cloned into vector pSilencer3.0-H1 between BamHⅠand HindⅢrestriction enzyme site.The recombinant plasmids pSilencer3.0-VEGF-C/siRNA1-3 and one negative control pSilencer3.0-VEGF-C/siRNA4 were obtained sequenced.Breast cancer cells MDA-MB-435 were transfected with the recombinant plasmid by positive liposomal transfection methods.The mRNA expression of VEGF-C was detected by Real Time-PCR and protein expression was detected by Western blot analysis.Our study showed that the recombinant plasmids containing VEGF-C siRNA were successfully constructed.The gene and protein expression of VEGF-C in breast cancer cells MDA-MB-435 were markedly decreased after transfection.The subgroup pSilencer3.0-VEGF-C/siRNA2 had the most powerful inhibitory effect.Compared to the negative control group,the gene expression of VEGF-C decreased 85.4%in the subgroup pSilencer3.0-VEGF-C/siRNA2 after transfection 48 hours,p＜0.0001.So we chose pSilencer3.0-VEGF-C/siRNA2 for next experiments.In vitro research,the influences on proliferation and apoptosis were studied by MTT and flow cytometry in the subgroup pSilencer3.0-VEGF-C/siRNA2.We also examined the gene expression of COX-2,CXCR4 by RT-PCR and protein expression of COX-2,Bcl-2 by Western blot to find the successive function of VEGF-C down-regulation.Our data showed that the cells apoptosis rate was 60%after transfection 72h,and cells grew much slower than control groups,p＜0.0001. It demonstrated that the down-regulation of VEGF-C can meantime inhibited the expression of some other genes such as COX-2,CXCR4 and bcl-2 which are important to tumor growth and metastasis.The results well explained the fuction of VEGF-C and the cross-talk of downstream tyrosine kinase activity.In vivo studies,breast cancer cells MDA-MBA-435 were injected subcutaneously into nude mice mammary glands to establish a successful transplant model of breast cancer.RNAi vectors were injected around the tumor.The volume of the transplant tumors were measured three times each week.After 4 weeks,mice were killed.The weighs of the transplant tumors were tested.In pathological examination,hematoxylin-eosin stain was performed to evaluate tumor growth.Pan-cytokeratin immunohistochemical stain was performed to examine lymphatic metastasis.VEGFR-3 and CD31 immunohistochemical stain was performed to examine microlymphatic density(MLD) and microvessel density(MVD) by 'hot spot' count.Transplant tumors in treated groups grew slowly,had smaller volume and lower weight compared to the control groups,p＜0.01.RT-PCR revealed VEGF-C gene expression down-regulation in treated groups. Immunohistochemical stain of lymphatic specific factor VEGFR-3 and vascular endothelial specific factor CD31 revealed that the control groups have higher MLD and MVD than treated groups.Also we observed much more lymphatic metastasis rate which was 60%in control groups than in treated groups which had none lymphatic metastasis.In summary,our study revealed that siRNA-mediated VEGF-C gene silencing was an effective tool to inhibit VEGF-C expression in human breast cancer cells in vitro and in vivo.The down-regulation of VEGF-C can inhibit proliferation,induce apoptosis,lessen lymphatic and vascular metastasis in breast cancer.The inhibition of lymphangiogenesis by VEGF-C RNA interference represents a new target for the development of anti-cancer gene therapies.