Protein 4.1R Suppresses VEGFA Secretion In M2 Macrophages To Inhibit Colon Cancer Metastasis

BackgroundColon cancer refers to a malignant tumor of the digestive tract that occurs in the colon.It is a high-grade malignant tumor in humans.Its incidence has been increasing.It has risen to the third place in the global incidence of malignant tumors and seriously harms human health.Tumor metastasis is one of the leading causes of death in patients with advanced colon cancer,and it is also a difficult problem in clinical treatment of colon cancer.Colon cancer metastasis means that colon cancer tumor cells invade the lymphatic vessels from the primary site,blood vessels or other pathways to other sites forming the same type of tumor as the primary site tumor.There are four ways of colon cancer metastasis,including direct infiltration,lymphatic metastasis,hematogenous metastasis and implant metastasis,lymphatic metastasis is the main way of colon cancer metastasis.The specific mechanism of colon cancer metastasis is still unclear,however,it has been reported that M2macrophages in the tumor microenvironment?TME?can promote tumor metastasis.Macrophages are derived from bone marrow pluripotent hematopoietic stem cells,which are lately differentiated cells with phagocytic function in the mononuclear-macrophage system and an important part of the body's innate immune defense line.Because of its heterogeneity,plasticity,and importance in maintaining the body's homeostasis,it has become an important content of medical immunology research.Macrophages can be divided into various subgroups according to their membrane surface antigens and secreted cytokines,mainly including classically activated M1 macrophages and selectively activated M2 macrophages.M1macrophages are believed to promote inflammatory responses and anti-tumor immune functions,while M2 macrophages show anti-inflammatory and tumor progression effects.Macrophages in the tumor microenvironment are called tumor-associated macrophages?TAMs?,which are the largest number of immune cells in the tumor microenvironment.The current mainstream view is that TAMs belong to the M2 activation pathway,and TAMs activation regulation plays an important role in the development and treatment of tumors.M2 macrophages secrete a variety of anti-inflammatory cytokines,immunosuppressive factors and tumor growth factors to promote angiogenesis,stromal decomposition and cancer cell movement,all of which are indispensable factors for tumor cell growth and metastasis.Therefore,M2 macrophages are a valuable cancer treatment target.However,the mechanism of M2 macrophages in tumor metastasis is still unclear.There may be a variety of molecules that play a regulatory role in it.Finding and identifying new regulatory molecules is of great significance for studying the function of M2 macrophages and cancer treatment.Protein 4.1R is a membrane skeleton protein component originally found in mature red blood cells.In human and mouse mature red blood cells,protein 4.1R is a bridge molecule that connects transmembrane proteins to the cytoskeleton network and plays an important role in maintaining the shape and membrane stability of red blood cells.Later,researchers found that the protein 4.1R encoding gene EPB41 is not only expressed in red blood cells,but also in various immune cells derived from bone marrow hematopoietic stem cells and played an important regulatory role.For example,recent reports have shown that in CD8⁺T cells,4.1R gene knockout significantly enhances IL-2 and IFN-?secretion levels and promotes their activation,in mast cells,4.1R can positively regulate Fcs RI signal transduction in vivo and in vitro.However,the role of protein 4.1R in M2 macrophages has not been reported so far.ObjectiveIn this study,the bone marrow-derived M2 macrophages from C57BL/6 mice of4.1R^+/+and 4.1R^-/-were used to investigate the functions of protein 4.1R on phagocytosis,cytokine secretion,and tumor-promoting effects of M2 macrophages.The results of the study are helpful to reveal the new function of protein 4.1R in M2macrophages.Methods1.The bone marrow cells of wild-type(4.1R^+/+)and 4.1R knock-out(4.1R^-/-)C57BL/6 mice were extracted,and differentiate into 4.1R^+/+M2 macrophages(M2-4.1R^+/+)and 4.1R^-/-M2 macrophages(M2-4.1R^-/-)induced by M-CSF and IL-4;The phenotype and purity of M2-4.1R^+/+and M2-4.1R^-/-were identified by flow cytometry.2.The expression of protein 4.1R in M2-4.1R^+/+and M2-4.1R^-/-was detected by PCR and Western blot.3.The effect of 4.1R gene knockout on the phagocytic function of M2macrophages was detected using a fluorescent microsphere method.4.The effect of protein 4.1R gene knockout on the expression of cytokines in M2 macrophages was detected by q RT-PCR;Westerrn blot and ELISA were used to detect the effect of protein 4.1R gene knockout on VEGFA secretion by M2macrophages.5.Co-culture M2-4.1R^+/+and M2-4.1R^-/-with mouse colon cancer cell MC38,respectively.Cell scratch test was used to detect the effects of protein 4.1R gene knockout on the ability of M2 macrophages to promote wound healing in MC38cells.Transwell test was used to detect the effects of protein 4.1R gene knockout on the ability of M2 macrophages to promote migration and invasion in MC38 cells.CCK-8 method was used to detect the effects of protein 4.1R gene knockout on the ability of M2 macrophages to promote proliferation in MC38 cells.Western blot was used to detect the effects of protein 4.1R gene knockout on the ability of M2macrophages to promote epithelial-mesenchymal transition?EMT?process in MC38cells.6.M2-4.1R^+/+and M2-4.1R^-/-were mixed with MC38 cells at a ratio of 1:2,and injected subcutaneously into the flank of BALB/c athymic nude mice,and the changes in tumor volume were observed and recorded.Finally,collect tumor tissue,take a picture and weigh it.7.After MC38 cells were treated with VEGFA,and VEGFA neutralizing antibodies were added to the co-culture system of M2-4.1R^-/-and MC38 cells,the effects of increased VEGFA secretion in M2-4.1R^-/-on the migration,invasion and EMT process of MC38 cells were detected by Transwell experiment and Western blot.8.After adding PI3K/Akt signaling pathway inhibitor LY294002 to the co-culture system of M2-4.1R^-/-and MC38 cells,the effects of increased VEGFA expression in M2-4.1R^-/-on the activation status of PI3K/Akt signaling pathway in MC38 cells were detected by Transwell experiment and Western blot.Results1.M2-4.1R^+/+and M2-4.1R^-/-were successfully induced,which laid the foundation for subsequent experiments.2.The results of PCR and Western blot showed that protein 4.1R was normally expressed in M2-4.1R^+/+,but was lost in M2-4.1R^-/-.3.Fluorescence microsphere detection showed that M2-4.1R^+/+phagocytosis was significantly higher than M2-4.1R^-/-.4.q RT-PCR,westerrn blot,and ELISA showed that VEGFA secretion in M2-4.1R^-/-was significantly higher than that in M2-4.1R^+/+.5.In vitro,after co-culturing M2-4.1R^+/+and M2-4.1R^-/-with MC38 cells,,the functions such as proliferation,migration,invasion and other functions were measured.The results showed that compared with M2-4.1R^+/+,the wound healing,migration,invasion,proliferation ability and EMT process of MC38 cells were significantly promoted after co-culture with M2-4.1R^-/-.6.In vivo,BALB/c athymic nude mice subcutaneous tumor transplantation models were used to detect the effects of M2-4.1R^+/+and M2-4.1R^-/-on the growth of MC38 tumors.The results showed that compared with M2-4.1R^+/+,MC38 cells mixed with M2-4.1R^-/-had the largest tumor formation and the fastest growth rate.7.VEGFA significantly enhanced the migration,invasion ability and EMT process of MC38 cells,and after neutralizing the VEGFA activity secreted in M2-4.1R^-/-,the migration,invasion ability and EMT process of MC38 cells were significantly inhibited.8.PI3K/Akt signaling pathway inhibitors significantly inhibited the migration and invasion ability and EMT process of MC38 cells co-cultured with M2-4.1R^-/-.Conclusions1.Protein 4.1R increased the phagocytosis of M2 macrophages.2.Protein 4.1R inhibited the tumor-promoting effects of M2 macrophages.3.Protein 4.1R down--regulates the secretion of VEGFA by M2 macrophages,inhibited the PI3K/Akt signaling pathway of colon cancer cells and inhibited their metastasis.