M1 Macrophages Inhibit NF-kB Signaling Pathway To Reverse The Epithelial Mesenchymal Transition Induced By M2 Macrophages In Bladder Cancer

Objectives:Bladder tumors are at the top of common tumors in the urinary system.Bladder tumor has the characteristics of high incidence,high recurrence rate and strong invasion,and its monitoring,diagnosis,treatment and other related costs are expensive,which brings heavy medical burden to patients and society.However,the mechanism of the occurrence and development of bladder cancer has not been fully elucidated.Lead to bladder malignant tumor biological behavior factors are diverse,tumor microenvironment is one of the important factors that induce bladder cancer enough,but the carcinogenic mechanism remains to be revealed.Epithelial-mesenchymal transformation is an important process in the development of epithelial-derived tumors.Tumor associated macrophage(TAM)play an important role in the tumor microenvironment,it contains two polarization each other subtypes: M1(classic activated macrophages)and M2 type(alternative activated macrophages).M1 TAM play an inhibit the growth of tumor,and M2 type TAM to promoting effect to the development of tumor.Based on the functional characteristics of two subtypes of tumor related macrophages,the current research focus on the relationship between macrophages and tumors will also be an important target for tumor therapy.The purpose of this experimental research is to explore the M2 macrophage stimulating Epithelial mesenchymal changes of the human bladder tumor cells(Epithelial and mesenchymal transition,EMT),and the nf-kappa B signaling pathways regulating role in this process,as well as M1 macrophages can inhibit the NF-kB signaling pathway to suppress the epithelium of the bladder tumor stroma.Methods Thp-1 was exposed at 150 nM PMA for 24 h and differentiated into M0 macrophages.On the basis of M0 macrophage,M0 macrophages were exposed to 24 h in medium containing 20 ng/ml IFN-and 10pg/ml LPS,and M1 type macrophages were obtained.On the basis of M0 macrophage,M0 macrophages were exposed to 24 h in the medium of 20 ng/ml il-4 and 20 ng/ml il-13,and the m2-type macrophages were obtained.The bladder tumor cells T24 and EJ were cultured in the lower compartment through 6 orifice small Chambers,and the M2 macrophages were planted in the upper chamber.The morphological changes of T24 and EJ cells were observed by inverted microscope.The wound healing assay results showed that the cell transfer ability was changed after the macrophages were cultured.Transwell test was used to detect changes in cell invasion ability.The protein and RNA content of epithelium were detected by Western blot and RT-PCR.Western blot test was used to detect the activity of NF-kam B signaling pathway.Then,we used the NF-kappa B pathway specific inhibitor PDTC and M1 macrophage and M1 macrophages to co-culture with bladder tumor cells to observe the morphological and molecular level changes of bladder tumor cells.Results(1)using PMA treatment THP 1 mononuclear cells after 24 h,mononuclear cells suspended into adherent cells,and grow pseudopodia,high expression of macrophage marker CD36,CD68,CD71,low expression of mononuclear cell markers CD14,M0 macrophages.The M0 macrophages were exposed to 24 h in medium containing 20 ng/ml IFN-and 10pg/ml LPS,and the M1 type macrophages were obtained,with high expression of il-6,il-1,TNF-,and cxcl-10.The M0 macrophages were exposed to 20 ng/ml il-4 and 20 ng/ml il-13 medium for 24 h,and the m2-type macrophages were obtained,with high expression of CD163,CD206,IL-10,and Arg-1.(2)M2 macrophages and bladder tumor cells EJ,T24 co-culture,tumor cells become scattered,cells from round or elliptic stretching into long fusiform,some of the cells are pseudopodia.The results showed that the healing ability of T24 cells was enhanced by the co-culture treatment of EJ.Transwell experiment demonstrated that EJ,T24 invasion ability was enhanced after the co-culture of M2 macrophages.The tumor cells of the bladder tumor treated by M2 macrophage showed an increase in the protein and mRNA expression of epithelial marker e-cadherin and zo-1,while the protein and mRNA expression of the interstitial marker vimentin and n-cadherind decreased.(3)the nf-kappa B pathway activation,under a certain number of M2 macrophages,as M2 macrophages concentration increases,the nf-kappa B protein specificity: p65,p50 nuclear expression levels,cytoplasmic phosphorylation of IKK alpha/beta(phosphorylated IKK alpha/beta)increases,and the pathway inhibitor I decreasing kappa B predominate.The protein test confirmed that,when the NF-kappa B pathway activity was effectively inhibited by the inhibitor PDTC,the m2-induced bladder tumor cells EJ and T24 epithelial mesenchymal were successfully reversed.Epithelial markers: expression of e-cadherin and zo-1 were up-regulated,and the expression of vimentin and n-cadherin was down-regulated.(4)M1 macrophages can inhibit the activity of NF-kappa B pathway,and achieve the effect of inhibiting m2-induced epithelial mesenchymal transformation.Through protein and gene detection,the expression level of epithelial protein was up-regulated,and the expression level of interstitial protein decreased.Conclusion : M2 macrophages can induce epithelial mesenchymal transformation of human bladder tumor cells,and NF-kappa B signaling pathway is regulating the interepithelial mesenchymal transformation of bladder tumor cells induced by M2 macrophages.M1 macrophages can inhibit m2-induced epithelial mesenchymal transformation,and M1 macrophages can inhibit NF-kappa B activity.These studies showed the nf-kappa B in the bladder tumor epithelial mesenchymal transformation caused by M2 macrophages process has an important regulation function,M1 macrophages can inhibit the carcinogenic process of M2 macrophages,can play the effect of inhibiting tumor occurrence and progress.