MiRNA Let-7b Regulates Macrophage Polarization And Its Effects On Prostate Cancer Cell PC-3

Objective: To investigate the role of let-7b in regulating macrophage polarization and its effect on human prostate cancer cell PC-3.Methods:1.Normal male human peripheral blood mononuclear cells were differentiated to M0,M1 and TAM.We transfected M1 and TAM in vitro differentiated macrophages with inhibitor or mimics of let-7b respectively;2.The morphology of macrophages was observed through an ordinary microscope and the expression of CD206 was determined by immunohistochemistry;3.The expression levels of IL-10,IL-12,IL-13,TNF-? and let-7b in the subtypes of macrophages were detected by q-PCR;4.The protein level of SCOS1 in such subtype of macrophage was detected by ELISA;5.The phosphorylation of STAT fanmily was analysised by Signal path chip;6.Then we carried out CCK8,wound healing,transwell assays and matrigel assay to detect the effects of the subtypes of macrophages on the proliferation,migration and angiogenesis of PC-3 respectively.Additionally,the ability of phagocytosis was tested by Staphylococcus aureus antigen.Results: 1.Through an ordinary microscope,M0 has a shape of long spindle,TAM with the inhibitor of let-7b was similar to M1,larger and the shape was plump,M1 with the mimics of let-7b was similar to TAM,the morphology was irregular,the pseudopodium and protuberance were longer;2.The effects of Let-7b on macrophage polarization: the expression level of CD206 in M0,M1 and TAM with the inhibitor of let-7b through fluorescence microscope was similar in the low level,in TAM and M1 with the mimics of let-7b was also similar at a high level;3.The effects of let-7b on the cytokines secreted by the subtypesof macrophages: The expression level of IL-10,IL-12,IL-13 and TNF-? were all low in M0 with the low expression level of let-7b;3.The high expression level of let-7b in TAM,M1 with the mimics of let-7b,The expression levels of IL-13,IL-10 and TNF-? were significantly increased,while the expression level of IL-12 is low;at the same time,comprared with TAM,M1 with the mimics of let-7b,the results were opposite in M1,TAM with the inhibitor of let-7b;4.The effect of let-7b on SOCS1 signaling pathway: the expression of SOCS1 in TAM and M1 with the mimics of let-7b with high expression level of let-7b was significantly higher than that in M0,M1 and TAM with the inhibitor of let-7b with low expression level of let-7b;5.The effect of let-7b on STAT signaling pathway: the protein phosphorylation levels of STAT1,STAT3,STAT5 a in TAM and M1 with the mimics of let-7b with high expression level of let-7b were significantly higher;6.The effects of let-7b on the functions of the subtypes of macrophages: TAM and M1 with the mimics of let-7b with high expression level of let-7b significantly promoted the proliferation,migration and angiogenesis on prostate cancer cell PC-3;7.The effect of let-7b on the phagocytic ability of the subtypes of macrophages: the ability of phagocytosis by TAM and M1 with the mimics of let-7b with high expression level of let-7b was obviously decreased.Conclusion:1.Let-7b has regulated the mutual polarization between M1 and TAM;2.Phosphorylation of STAT1,STAT3 and STAT5 a in TAM may be related to the negative regulation of SOCS1 by let-7b;3.Let-7b regulats SOCS1/STAT signal pathway to change the expression of the cytokines IL-10,IL-12,IL-13,TNF-?;4.TAM can significantly promote proliferation,invasion,migration and angiogenesis on PC-3,the mechanism may be related to the singnal pathway of SOCS1/STAT were regulated by let-7b,which influences the expression of cytokines in different subtypes;5.The mutual polarization between M1 and TAM by let-7b decreased the immune level of prostate cancer patients after operation.