Effects Of MicroRNA-22 On Macrophages And Therapeutic Efficacy Against Glioma Through Regulating Macrophages

Glioma is the most common primary tumor in the brain,accounting for about 81%of the malignancies in the central nervous system（CNS）.They usually originate from glia or progenitor cells and progress to astrocytoma,oligodendroglioma,ependymoma,or oligodendroglioma.According to the classification of the World Health Organization,glioma are divided into four grades,among which grade Ⅰ and Ⅱ gliomas are low-grade glioma and grade Ⅲ and Ⅳ glioma are high-grade glioma.In general,relatively high grades are associated with a poor prognosis.Current treatments including maximum safe surgical resection,and combined with radiotherapy and temozolomide chemotherapy and other comprehensive treatments.Although surgery can relieve clinical symptoms and prolong survival,due to the infiltration of tumors,the prognosis of patients with high-grade glioma is still poor,newly diagnosed cases of 5-year survival rate less than 15%,and the median survival time is about 15 months.It is gradually recognized that the development of solid tumors in a complex microenvironment,which seriously affects tumor growth,transformation and metastasis.In the microenvironment of most solid tumors,there are a variety of non-neoplastic cell types,including fibroblasts,immune system cells,and endothelial cells.Each of these stromal cells produces growth factors,chemokines,extracellular matrix components,and angiogenic molecules that can alter the local environment in which tumor cells grow and infiltrate.In the case of glioma,macrophages are the primary source of these stromal factors.In addition,macrophages are the largest population of immune cells in glioma and a key component of innate immunity,capturing and processing antigens from tumor cells and presenting them to T cells of the adaptive immune system.Macrophage-mediated tumor phagocytosis and subsequent antigen presentation are critical for generating anti-tumor immunity and increase the possibility of targeting macrophages as adjuvant therapies for glioma.MicroRNAs（miRNAs）are non-coding molecules involved in the regulation of posttranscriptional genes,which have been shown to regulate the proliferation and apoptosis of tumor cells and act as oncogenes or tumor suppressor genes.miR-22 has been shown to participate in a variety of biological processes,and has the function of antitumor in a variety of tumors including glioma.Recent studies have shown that miR-22 involved inflammatory immune response in macrophages and T cells,suggesting that miR-22 may affect the growth,transformation and metastasis of glioma by regulating macrophages in the tumor environment.Therefore,this study mainly discusses whether miR-22 can regulate macrophages to affect the development of glioma and its mechanism.Objective:1.This study aimed to determine whether miR-22 can regulate macrophages and affect the development of glioma.2.This study aims to determine whether miR-22 can regulate macrophage-mediated T cell priming,and to reveal the potential clinical value and molecular mechanism of miR-22 in regulating glioma tumor microenvironment.Methods:1.Real-time quantitative polymerase chain reaction is used to detect the expression of miR-22 in glioma tissue samples and macrophages in tumors.2.GL261 situ glioma model was used to investigate the tumor suppressive effect of miR-22 in vivo.The survival cycles of homozygous(miR-22^-/-),heterozygous(miR-22^+/-)and wild type(miR-22^+/+)mice were recorded.Tissues were stained with H&E;immunohistochemistry was used to detect the expression of PCNA,F4/80 and MHCⅡin tumor tissue;The proportion of CD11b⁺macrophages in tumor tissues was detected by flow cytometry.3.Primary macrophages and primary glioma cells isolated from glioma tumor,and combined with RAW264.7,THP-1-M,U251,U-87 MG,and GL261 to construct co-culture system,which to detect glioma cell proliferation and migration.To investigate whether miR-22 can regulate macrophages and thus affect the proliferation and migration of glioma,and to verify the effect of miR-22 on the phenotype of macrophages by detecting the expression of TNFα,IL-2 and IL-12 in macrophages by ELISA assay.4.RNA sequencing revealed the specific functions of miR-22 and miR-22-related pathways in macrophages.Flow cytometry was performed to prove the phagocytosis of miR-22 in RAW264.7 cells and primary macrophages.RAW264.7 cells and GL261cells,THP-1-M cells and U251 and U-87 MG cells were directly mixed and co-cultured,and flow cytometry was used to detect whether miR-22 can enhance the ability of macrophages to phagocytose glioma cells.To investigate the dynamics of tumor cell phagocytosis via macrophages,representative still images of GL261 uptake by RAW264.7 was showed in a live-cell imaging experiment.5.In order to determine whether the enhancement of phagocytic ability will also enhance the antigen presentation in macrophages,glioma cells were co-cultured with macrophages,and the effects of miR-22 on antigen presentation related molecules MHCII and costimulatory molecule CD80 on macrophages were detected by flow cytometry.6.The effect of miR-22 on the nuclear translocation of phosphorylation p65 in macrophage co-cultured with glioma cells was detected by imaging flow cytometry.And the expression of CD11b and p-P65 in tumor tissues of glioma model was detected by immunofluorescence.7.To test whether miR-22-overexpressing macrophages can promote the activation of CD8⁺T cells.Therefore,human CD3⁺T cells and mouse CD3⁺T cells were selected from glioma tissues and mice spleen by flow cytometry,and CD3⁺T cells were added into the co-culture of glioma cells and macrophages transfected with miR-22 mimics.The proportion of CD8⁺effector T cells producing IFNγwas detected by flow cytometry.8.Western blot was used to detect the HDAC6 protein changes in macrophages transfected with miR-22 mimics,and luciferase reporter verified that HDAC6 is the target of miR-22.Immunohistochemistry was used to detect the expression of HDAC6in glioma tissues and adjacent tissues,and immunofluorescence was used to detect the expression of CD11b and HDAC6 in glioma tissues and adjacent tissues.To test whether macrophages treated with two selective HDAC6 inhibitors,Tubacin and CAY10603 could mediate the biological effects of miR-22 in macrophages.Results:1.The expression of miR-22 in glioma tissues and macrophages.The expression of miR-22 in glioma tissues and adjacent tissues was detected by q RT-PCR.Compared with adjacent tissues,miR-22 expression was significantly reduced in glioma tissues.Consistently,the expression of miR-22 in primary macrophages sorted from glioma tissues was also dramatically lower than that inadjacent tissues.2.In the orthotopic glioma model,the effect of miR-22 knockout on tumor formation and macrophage infiltration.In the orthotopic glioma model,the knockout of miR-22 reduced the survival times of mice and promoted the growth of tumors.Histopathological examination of tumor tissues by H&E staining showed that neonatal tumor tissue was abundantly increased in miR-22-knockout mice compared to that in wild-type groups.Tissue analyses further suggested a marked increase in PCNA⁺proliferating tumor cells in miR-22^-/-mice.Flow cytometry was used to detect the proportion of CD11b⁺macrophages in tumor tissues at a relatively early stage of tumor development（7 days after injection of glioma cells）.Glioma tissues from miR-22^-/-mice exhibited a remarkable decrease in CD11b⁺macrophages compared to that in wild-type controls and the immunohistochemical results showed a decrease in F4/80⁺or MHCⅡ⁺activated macrophages in miR-22^-/-mousetumor tissues.3.The effect of miR-22 on the function of glioma cells by regulating macrophages.Primary macrophages and primary glioma cells isolated from glioma tumor,and combined with RAW264.7,THP-1-M,U251,U-87 MG,and GL261 to construct co-culture system,which to detect glioma cell proliferation and migration.To investigate whether miR-22 can regulate macrophages and thus affect the proliferation and migration of glioma,and to verify the effect of miR-22 on the phenotype of macrophages by detecting the expression of TNFα,IL-2 and IL-12 in macrophages by ELISA assay.CCK-8 and Transwell experiments confirmed that miR-22 could indirectly inhibit the proliferation and migration of tumor cells through macrophages.ELISA confirmed that miR-22 increased the expression levels of TNFα,IL-2 and IL-12in macrophages.4.The effect of miR-22 on the phagocytic ability of macrophages.RNA sequencing revealed that miR-22 is closely related to the phagocytic function of macrophages.Flow cytometry proved that miR-22 stimulated the phagocytosis of RAW264.7 and primary macrophages.RAW264.7 and GL261,THP-1-M and U251,U-87 MG were directly mixed and co-cultured,in which macrophages were labeled with CD11b and tumor cells were labeled with CFSE.The imaging flow cytometry results confirmed that miR-22 enhanced the ability of macrophages to phagocytose glioma cells.5.The effect of miR-22 on antigen presentation related molecules and p-P65 in macrophages.In order to determine whether the enhancement of phagocytic ability will also enhance the antigen presentation in macrophages,glioma cells were co-cultured with macrophages.Flow cytometry was used to confirm that miR-22 increased antigen presentation related molecules MHCII and costimulatory molecule CD80,and increases the nuclear translocation of NF-κB phosphorylated p65.As well as decreased nuclear translocation and phosphorylation of the NF-κB p65 in macrophages in miR-22-knockout glioma-bearing mice in vivo.6.The effect of miR-22-overexpressing macrophages on T cell activationCo-cultured macrophages transfected with the miR-22 mimics and the NC mimics with glioma cells were subsequently exposed to CD3⁺T cells.The amount of interferon gamma（IFNγ）-producing CD8⁺effector T cells obviously increasedin macrophages transfected with the miR-22 mimics.Macrophages treated with a NF-κB inhibitor not only showed abrogated nuclear translocation of p65 and phosphorylation but also significantly inhibited MHC II and CD80 expression and CD8⁺T cell-priming effects.7.Targeting relationship between miR-22 and HDAC6Western blotting results showed that miR-22suppressed HDAC6protein level expression in RAW264.7 cells,and luciferase reporter gene detection verified that HDAC6 is the target of miR-22.Macrophages treated with HDAC6 inhibitor increased the expression levels of TNFα,IL-2 and IL-12 in the culture medium,and inhibited the proliferation and migration of glioma cells.The expression of HDAC6 was higher in gliomas than in normal tissues,and consistent results were also observed in macrophages,which is in contrast to the expression of miR-22.In addition,Tubacin and CAY10603significantly boosted the phagocytic ability of macrophages in a dose-dependent manner.Similarly,an increased glioma cell phagocytosis by HDAC6-deficient macrophages was observed.Similar to that with miR-22-overexpressing macrophages,HDAC6-deficient macrophages showed enhanced nuclear translocation and phosphorylation of p65,antigen presentation,and CD8⁺T cell-priming effects.Conclusions:1.Compared with adjacent tissues,the expression of miR-22 is reduced in glioma tissues.Consistently,the expression of miR-22 in primary macrophages sorted from glioma tissues was also dramatically lower than that inadjacent tissues.2.In the orthotopic glioma model,knockout of miR-22 promoted tumor growth and reduced macrophage infiltration at a relatively early stage.3.miR-22 increases the expression of TNFα,IL-2 and IL-12 in macrophages,and inhibits the proliferation and migration of glioma cells by regulating macrophages.4.miR-22 increases the phagocytic capacity of macrophages and increases the expression of antigen presentation related molecules and p-P65 nuclear translocation.5.miR-22-overexpressing macrophages increases antigen presentation and CD8⁺T cell activation in an NF-κB-dependent manner.6.HDAC6 inhibition can mediate the anti-tumor effect of miR-22 overexpression in macrophages,and HDAC6 is a functional target of miR-22.