The Effect Of SiRNA Targeting Survivin And VEGF-C On Mouse Breast Cancer And The Effect Of M2 Macrophage On 4T1 Cell Metastasis

Part 1.Downregulation of survivin expression exerts antitumoral effects on mouse breast cancer cells in vitro and in vivoBACKGROUND & OBJECTIVE: In order to elucidate the role of survivin in breast cancer metastasis,short interfering RNA(siRNA)was used in the present study to specifcally downregulate survivin expression in the murine breast cancer cell 4T1 and explore the antitumor effect of survivin-siRNA in vitro and in vivo..METHODS: 1.Cell experiment:Three pairs of siRNA targeting mouse survivin were designed and chemically synthesized.The most effective one was selected by analysis of RT-PCR and Western blot;survivin-siRNA was transfected into 4T1 cells;proliferation,migration and apoptosis of 4T1 were detected by MTT assay,flow cytometry,RT-PCR,Western blotting and other methods to analyze the effect of survivin-siRNA.2.Animal experiment:Balb/c mice bearing breast cancer was used to investigate the effect of down-regulating survivin on tumor growth and metastasis.Immunohistochemical assay for podoplanin protein expression and lymphatic microvessel density in tumor tissue were performed to explore the anti-tumor mechanism of down-regulating survivin on tumor microenvironment.RESULTS: 1.Cell experiment: RT-PCR and Western blot results showed that si S3 was the most efficient of three pairs of siRNA transfected to 4T1 cells.48 hours after 4T1 cells transfected with survivin-siRNA,RT-PCR and Western blot showed that the m RNA and protein expression of survivin,p AKT,HIF-? and VEGF-C were significantly decreased;MTT showed that proliferation of 4T1 cells significantly reduced;cell migration experiments showed that migration and invasion abilities of 4T1 cell significantly decreased.2.Animal experiment: When injected intratumor,the survivin-siRNA significantly inhibited the growth of orthotopically transplanted 4T1 tumors in vivo.In addition,the number of pulmonary metastases and the microlymphatic vessel density were significantly reduced.CONCLUSION: The results demonstrated that down-regulating expression of survivin by siRNA effectively inhibited the proliferation,migration and invasion abilities of murine breast cancer cells in vitro;could inhibit tumor growth and lung metastasis,and reduce the microlymphatic vessel density in vivo.Part 2.M2 macrophage promoted breast cancer 4T1 cell to metastate,co-silencing VEGF-C and survivin inhibitd solid tumor growth of murine mammary carcinomaBACKGROUND & OBJECTIVE: To explore the effect of mouse macrophages with different activated phenotype on the proliferation and invasion of mouse brast cancer cells.METHODS: 1.Cell experiment:Macrophages were treated by mr IFN-? + LPS or mr IL-4 for 24 h respectively,then identified by q RT-PCR to detect i NOS the marker of classically activated macrophages and Fizz1-the marker of alternatively activated macrophages.Based on the co-culture Transwell systems of activated macrophages and 4T1 cells,cell growth curves of 4T1 cells were drawn,vascular endothelial growth factor-C(VEGF-C)expressions of 4T1 cells were detected by ELISA,and NO concentrations of co-culture fluid supernatant were determined by nitrate reductase method.2.Animal experiment:inhibitory effect of siRNAs on xenograft tumors and lung metastases was observed in balb/c mice.RESULTS: 1.Cell experiment : Mouse M1 and M2 macrophags models were established successfully.Among the co-culture groups,the growth of 4T1 cells and VEGF-C expression were the fastest and strongest in M2 group;NO concentration was high in M1 group.The proliferation and invasion of 4T1 cells were promoted mostly in M2 group,but inhibited in M1 group.2.Cell experiment : siRNA can inhibit tumor-bearing mice transplanted tumor growth and lung metastasis.Tumor-associated macrophages compared with spleen cells express higher levels of CD206.CONCLUSION: M2 macrophages promote 4T1 cell proliferation,migration and invasion by the promotion of VEGF-C expression.Co-siRNAs down-regulating VEGF-C and survivin in tumor-bearing mice showed good anti-tumor and anti-metastasis effect.Tumor-associated macrophages in tumor-bearing mice might be polarized to the alternative activation phenotype(M2).