| Objective Angiogenesis and immunosuppression are the main biological mechanisms responsible for cancer progression.It has been reported that immune deficiency or impairment is a common phenomenon in patients with cancer,and tumor cells have developed a variety of cellular and molecular mechanisms to escape from antitumor immune responses.Dendritic cells(DC) play a central role in antitumor immune responses.Abnormal differentiation of DC and its inability to stimulate T cells are the important factors in tumor escaping from immune-system supervision. Vascular endothelial growth factor(VEGF) is the most important pro-angiogenic factor,produced by many human malignancies.Recent research has shown that VEGF may also play an immunosuppressant role by inhibiting the maturation of DC from hematopoietic progenitors in addition to its angiogenic activity.Based on VEGF dual roles of angiogenic activity and immune inhibition,we expected therapeutic blockade of VEGF action using small interfering RNA might inhibit cancer angiogenesis as well as improve prospects for immunotherapy.In this study,we observed the effect of dendritic cells loaded tumor antigen combinated siRNA targeting VEGF gene on MCF-7 cells,investigated whether this combination could improve anticancer immunity in vitro to provide the experimental foundation for combination of antiangiogenic and immunotherapy in cancer.Medhods Small interfering RNA(siRNA) targeting VEGF gene was designed and synthesized,then transfected cells with Lipofectimine to observe the effect of siRNA on VEGF gene and MCF-7 cells;The peripheral blood were drawn from healthy adult persons,then cultured DC with granulocyte-macrophage colony stimulating factor(GM-CSF),interleukin 4(IL-4) and tumor necrosis factor-a(TNF-a).Mononuclear cells were cultured with the culture supernatants from primary MCF-7 cells and cytokines to analyse the effect of the culture supernatants on the differentiation,maturation and function of DCs;Then siRNA directed against VEGF gene was designed and transfected into MCF-7 cells,so as to invested the change of the effect of the culture supernatants on the differentiation, maturation and function of DCs;Dendritic cells loaded tumor antigen combinated siRNA targeting VEGF gene on MCF-7 cells,from which we could investigate whether this combination could improve anticancer immunity.Results The siRNA targeting human VEGF effectively silenced VEGF gene and inhibited the secretion of VEGF in MCF-7,whereas the control scramble siRNA showed no effects;MCF-7 cell supernatant could inhibit the differentiation,maturation and function of DC induced from PBMC,the cultured DC cultured with MCF-7 cell supernatant showed low expression of CD80,CD83,CD86 and HLA-DR,and IL-12 secreted by DC and IFN-γproduced by PBMNC all reduced(p＜0.01);After silenced VEGF gene DCs cultured in the culture supernatants from MCF-7 cells had significantly higher expression of CD86,CD80,CD83 and HLA-DR than the control group(p＜0.01),IL-12 secreted by DCs and IFN-γproduced by PBMNC were improved significantly(p＜0.01);The activity of antitumor of dendritic cells loaded tumor antigen combinated siRNA targeting VEGF gene was markly,the target cells were lysised,and the inhibition rate was 99%.Conclutions siRNA designed and synthesized in vitro could reduce VEGF gene expression, inhibit proliferation and induce apoptosis of MCF-7 cells;the culture supernatants from MCF-7 breast cancer cells could inhibit the development and functions of DCs;siRNA specifically inhibited VEGF expression in MCF-7 cells,the inhibition of DC maturation and function in culture supernatants from siRNA transfected cells was significantly decreased.It suggested that VEGF might play an important role in tumor developmemt,progress and immunorepression;Dendritic cells loaded tumor antigen combinated siRNA targeting VEGF gene could markedly inhibit the proliferation of MCF-7 cells,this provided rationale for breast cancer clinical practice.
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