The Mechanism Of MiR-99b-5p-mediated Notch Signaling On Regulating Macrophage Activation And Its Therapeutic Role By Targeting TAMs In Tumor Progression

?Background?Tumor has seriously threatened human health.Because it owns the features including invasive growth,metastasis,resistance to apoptosis signals and evading immunologic cytotoxicity,which lead to hardly be cured by surgery,radiation therapy and chemotherapy.Therefore,to explore more effective therapeutic strategies become particularly significant.The carcinogenesis may be ascribed to not only tumor cells themselves,but also the inflammatory microenvironment of tumor,also known as tumor related inflammation,which plays a critical role in tumor initation and progression.Tumor associated macrophages(TAMs),also called M2-like macrophages,as one of the main infiltrated immune cells in tumor,play an important role in regulating tumor related inflammation.A large number of clinical studies have shown that increasing TAMs in tumor is closely associated with the poor prognosis of tumor patients.Notably,considering the role of TAMs in the development of tumor,many research groups demonstrate that inhibiting the number and function of TAMs can effectively hamper tumor growth and prolong the survival period of patients.In addition,in tumor microenvironment,macrophages exhibit two kind of activation model,one is the classical activated macrophages,namely M1 macrophages,which exert the role of anti-tumor activity.Another is the alternative activated macrophages,namely M2 macropahges which possess the role of pro-tumor activity.Therefore,some studies have shown that converting the activation pattern of TAMs and promoting their anti-tumor effects may impede tumor growth.However,the underlying mechanism on regulating the macrophage activation and function still remain unclear.Notch signal is a highly conservative signaling pathway,which regulates cell differentiation and development through the interaction of Notch ligands and receptors among adjacent cells.Once Notch signaling is activated by its ligands from neighboring cells,Notch intracellular domain(NICD),cleaved by?-secretase(GSI),is released and enters into the nucleus recruiting some activators and then combines the recombination signal-binding protein Jk(RBP-J),which is the key transcription factor of Notch signaling,to further regulate the transcription of Hes and Hey which determine the cell differentiation,development,proliferation,apoptosis and so on.Previous studies have proven that Notch signaling plays an essential role in not only the differentiation of T and B lymphocytes,but also the fate determination of monocytes to macrophages and dendritic cells(DCs).Our previous studies have verified that Notch signaling can regulate macrophage activation and function through several signaling pathways,including SOCS3,ERK1/2,AKT and CYLD/NF-?B signaling.Meanwhile,we find that Notch signaling may memory the macrophage polarization status.Thus,using microarray assay,we screened a series of downstream miRNAs of Notch signaling,including miR-125a,miR-148a and miR-99b-5p,from Notch signaling deficient BMDMs after LPS stimulation.Further studies demonstrate that miR-125a or miR-148a-mediating Notch signaling can promote macrophage M1 polarization while inhibit M2 polarization,resulting in the suppression of tumor growth and the enhanced capacity of engulfing bacteria.Because miR-99b-5p and miR-125a locate the promoter region of same host gene,we propose that miR-99b-5p may also regulate macrophage activation and function depending on Notch signaling.Delivery microRNAs into TAMs in tumor,such as miR-99b-5p,may be new strategy to treat tumor in future.?Objectives?1.miR-99b-5p is a new downstream molecule of Notch signaling on regulating macrophage activation and function.2.The mechanisms of miR-99b-5p-mediated Notch signaling on regulating macrophage activation and function;3.The role of miR-99b-5p and/or miR-125a in regulating TAMs activation for tumor therapy.?Methods?1.The method of culture and polarization of bone marrow-derived macrophage(BMDM)are established;2.The miRNA expression in Notch overexpression or knockout BMDM is detected by qRT-PCR;3.Reporter assay,real-time quantitative PCR and Western Blot are used to confirm the target genes of miRNA;4.Real-time quantitative PCR,Western Blot and FACS assay are used to investigate the role of miR-99b-5p in regulating macrophage activation and function;5.To construct the delivery system of microRNA into TAMs in tumor.Immunofluorescent staining and animal live imaging system are used to examine the targeting TAMs ability of the microRNA delivering system;6.In the othotopic hepatoma model,flow cytometry assay and immunofluorescence staining are used to analyze the TAMs phenotype and function,T lymphocyte subgroups,the apoptosis of tumor cells as well as the effects on the tumor progression.?Results?1.Using specific Notch signaling activated or blockade macrophages,the results show that Notch signaling can effectively regulate the expression of miR-99b-5p;2.Using real-time quantitative PCR and Western Blot,our result show that miR-99b-5p-mediated Notch signaling can promote macrophage M1 polarization,the expression of M1 markers,such as IL-12,TNF-a and IL-6,increased.Meanwhile,it inhibits M2 polarization,the expression of M2 markers,such as IL-10,MR,Arg1,decreased;3.Using flow cytometry assay,miR-99b-5p significantly promotes the MHCII expression in macrophages,consequently promotes CD4~+T cell proliferation,suggesting that miR-99b-5p can promote the antigen presenting ability of macrophages;4.Using report assay,real-time quantitative PCR and Western Blot,our result show that miR-99b-5p can result in the activation of NF-Nkiras2,thus promoting M1 macrophage polarization;while miR-99b-5p can inhibit M2polarization of macrophages by hampering mTOR/IRF4 axis;5.Using the experiments of immunofluorescence staining and animal live imaging system,our results show that the miRNA delivery system can target TAMs precisely;6.Using flow cytometry assay and immunofluorescence staining,our result show that the miR-99b-5p or miR-125a delivery system with targeting TAMs can reduce tumor growth by promoting M1 activation of TAMs,reversing immunosuppression T cell subgroups and inducing tumor cell apoptosis.?Conclusions?Taken together,our study demonstrates that miR-99b-5p is a new downstream molecule of Notch signaling.On one hand,miR-99b-5p can activate NF-?B signaling by inhibiting mTOR and Nkiras2 in macrophage,thus promoting M1 macrophage polarization.On another hand,miR-99b-5p inhibits M2 polarization of macrophages by inhibiting mTOR/IRF4 axis.The delivery of miR-99b-5p and/or miR-125a by targeting TAMs in liver cancer can inhibit the progress of liver cancer via promoting M1polarization of TAMs,reversing immunosuppression T cell subgroups and inducing the apoptosis of tumor cells.These results may be helpful to understand the epigenetic mechanism of Notch signaling on regulating TAMs activation through microRNA,and provide potential targets and strategies for tumor immunotherapy.