The Influence Of SiRNA Targeting VEGF-C In Mouse Breast Tumor Microenvironment

Purpose : The tumor microenvironment of breast cancer is the material basis of proliferation,invasion and metastasis for tumor cells.Vascular endothelial growth factor C(VEGF-C)in tumor microenvironment may induce lymphangiogenesis,metastasis and immune tolerance.however,the mechanism and relations between VEGF-C and breast tumor microenvironment has not yet been entirely cleared.In this study,we make 4T1 and DC as a starting point,interfering VEGF-C expression by RNAi technology,from molecular,cellular and whole animal level,to explore the relations and mechanism between VEGF-C and tumor microenvironment by intro and in vivo test.METHODS:1.In vitro experiments: design and chemical synthesis 3 pairs of siRNA targeting mouse VEGF-C,then choose the most efficent one by RT-PCR Western blot methods;cultured and transfected 4T1 cells and mouse dendritic cells with si V2;then detected the influence to proliferation,migration and apoptosis of 4T1 and the phenotype of DC by MTT assay,Hochest33258 staining,flow cytometry,RT-PCR,Western blotting and other methods.2.In vivo experiments: Balb/c mouse were inoculated with 4T1 cells to establish tumorbearing animal models for detecting the influence of VEGF-C interference on tumor growth and metastasis.TUNEL staining for tumor apoptosis,ELISA assay for tumor tissue VEGF-C and CCL21 expression,flow cytometry for tumor infiltrating DC and Treg conditions,immunohistochemical assay for VEGF-C,p AKT,KLF-2,Survivin and VEGFR-3 protein expression and lymphatic microvessel density in tumor tissue were performed to explore the anti-tumor effect and the mechanism of VEGF-C interference impacting on tumor microenvironment.RESULTS: 1.In vitro experiment:RT-PCR and Western blot results showed that si V2 was the most efficient of three pairs of siRNA transfected to 4T1 cells.48 hours after 4T1 cells transfected with 100 n M si V2,RT-PCR and Western blot showed that VEGF-C,VEGFR-3,p AKT,KLF-2 and Survivin m RNA and protein expression were significantly decreased and the activated Caspase-3 protein expression increased significantly;ELISA results showed that concentrations of VEGF-C and CCL21 in 4T1 supernatant were significantly reduced;MTT showed significantly proliferation reduce;Hochest33258 staining showed typical apoptosismorphological changes of 4T1 cells;flow cytometry showed that 4T1 cells apoptosis increasing significantly and CCR7 expression was significantly downregulated;cell migration experiments show 4T1 cell migration is significantly reduced.48 hours after mouse dendritic cells was transfected with si V2,ELISA showed that VEGF-C concentrations in mouse DC culture supernatant was significantly reduced;flow cytometry showed that mouse DC apoptosis was significantly increased;RT-PCR and Western blot showed that VEGF-C,VEGFR-3,p AKT,KLF-2 and Survivin expression in mouse DC were significantly reduced but activated Caspase-3 protein expression was significantly increased.48 hours after LPS stimulation,flow cytometry showed that on si V2 transfected murine DC,CD86,I-Ad and CCR7 expressionwere significantly lower than the control group,while there was no obvious difference in CD80 expression;MLR showed that the lymphocytes stimulating ability of si V2 transfected mouse DC reduced significantly.2.In vivo experiment:Animal experiments showed that si V2 injections inhibited tumor growth and lung metastasis;TUNEL staining showed apoptotic cells in tumor tissues was significantly increased;Immunohistochemistry showed that VEGF-C,p AKT,KLF-2,Survivin and VEGFR protein expression and lymphatic microvessel density in tumor tissue were significantly reduced;ELISA results showed that the expression of VEGF-C and CCL21 in tumor tissue reduced significantly;flow cytometry showed that the number of tumor infiltrating dendritic cells and Treg cells significantly reduced,the expression of CCR7 on infiltrating dendritic cells was significantly decreased.There were no significant difference of CD80,CD86 and I-Ad expression on infiltrating dendritic cells between the groups,and all the expression levels were less than mature DC.CONCLUSION: VEGF-C interference in 4T1 cells could reduce CCR7 and CCL21 expression,inhibite cells proliferation and increase apoptosis;VEGF-C interference in mouse DC could inhibit maturation,promote apoptosis and reduce the CCR7 expression;VEGF-C interference could inhibit tumor growth and lung metastasis,and reduce the lymphatic microvessel density and the number of Treg and naive DC in tumor microenvironment.The mechanism of the action may be related to VEGF-C expression silence,VEGFR-3 expression decreased,phosphorylation of AKT in PI3K/AKT signaling pathway decreased,inhibitor of apoptosis protein survivin and transcription factor KLF-2 downregulation.SIGNIFICANCE: this research design and screen a pair of effective siRNA against mice VEGF.Holding 4T1 cells and mice DC cells as the breakthrough point,we investigate the effect and mechanismof VEGF-C silence on the biological character of breast cancer cells and tumor microenvironment,in vitro and in vivio.This study lays a foundation for the clinical application of breast cancer.gene therapy.