Mechanism That Glioma Eliminates Tumor-suppressive MiRNAs Via Exosomes To Promote The Formation Immune Suppressive Microenvironment

BackgroundGlioblastoma(GBM)is the most common primary malignant tumor in the central system.Although great progress has been made in the comprehensive treatment of GBM recently,the overall prognosis remains poor and the long-term survival rate is still low.Compared with other solid tumors,the unique blood-brain barrier and immune microenvironment of GBM are great challenges to be faced during the development of new therapies for it,which is an important factor for its poor treatment outcome.Compared to other solid tumors,the tumor microenvironment(TME)of glioma is more complex and highly immunosuppressive due to the presence of the blood-brain barrier and its unique regional immune characteristics.Under physiological conditions,immune cells from the blood circulation are rarely present in the brain parenchyma due to the presence of the blood-brain barrier.When a tumor is present,various types of immune cells can migrate to the tumor area through the damaged blood-brain barrier and either exert anti-tumor effects or be influenced by the tumor cells to promote immunosuppression.Therefore,systematically elucidating the regional immunological characteristics of tissue during the development of glioma will provide an important theoretical basis for the diagnosis and treatment of the disease.It is well known that exosomes are key mediators of intercellular communication and are involved in various physiological and pathological processes,Exosomes contain proteins,lipids,nucleic acids and other substances,which can mediate the information transmission between donor cells and recipient cells to maintain the homeostasis against stress response.On the one hand,glioma can transfer its contents to other tumor cells through exosomes to promote their proliferation or invasion and migration.On the other hand,the contents can be delivered to immune cells,which can be modified to promote the immunosuppressive ability of the tumor microenvironment and promote the malignant progression of tumors.In order to comprehensively explore the regulatory mechanism of glioma exosomal miRNAs on tumor cells and their immune microenvironment,we performed whole transcriptome sequencing of CSF exosomes and matched tumor tissue samples from 59 patients.According to the distribution characteristics of miRNAs in glioma tissues and their exosomes,they were divided into three categories and five subtypes,namely exosomal type(high-selective sEV type,HSE and low-selective sEV type,LSE),intracellular type(high-selective cell type,HSC and low-selective cell type,LSC)and mixed type(MIX).The representative miRNAs of HSE and HSC were further studied.We found that glioma could promote the malignant progression by expelling tumor suppressor miRNAs from the cell;On the other hand,these tumor suppressor miRNAs can also modify the tumor immune microenvironment and promote the transformation of myeloid immune cells to immunosuppressive phenotype,thus achieving the dual effect on promoting the malignant progression of glioma.Part I Mechanism that glioma eliminates tumor-suppressive miRNAs via exosomes to promote the malignant progressionObjectivesTo investigate the classification of miRNAs in cerebrospinal fluid exosomes of glioma patients and the mechanism of glioma cells sorting miRNAs into exosomes.Methods1.Cerebrospinal fluid exosomes and tumor tissues from glioma patients were collected for sequencing,and the miRNAs highly expressed in glioma exosomes and tumor tissues were screened and classified.2.Select the representative miRNAs of exosomal and cellular types,overexpress them in glioma cells,and verify their effects on glioma cells by phenotypic experiments such as CCK8 and EdU.3.Use starbase and other databases to predict miRNA target genes,and validate them by knockdown and overexpression in glioma cells.4.For exosomal miRNAs,we screened the sorting proteins that sorted them into exosomes by RNA pull-down assay and other experiments,and verified the binding sequences to further investigate the mechanism.5.The role of miRNAs in glioma cells was further validated by in vivo experiments in mice.Results1.By analyzing the sequencing results of tumor tissues and cerebrospinal fluid exosomes from glioma patients,miRNAs were classified into three major categories and five subtypes according to their secretion characteristics,namely exosomal(exosomal high selective HSE and exosomal low selective LSE),intracellular(intracellular high selective HSC and intracellular low selective LSC)and mixed(MIX).2.miR-1298-5p is highly expressed in cerebrospinal fluid exosomes of glioma patients and can inhibit malignant progression of glioma.miR-9-5p is less expressed in cerebrospinal fluid exosomes of glioma patients than in tumor tissues and can promote malignant progression of glioma.3.miR-1298-5p can act by targeting SetD7.4.Glioma cells mediated miR-1298-5p into exosomes and excreted it out of cells via hnRNPA2B1,thus promoting the malignant progression of glioma.Conclusion1.Glioma cells promote malignant progression of glioma by sorting oncogenic miRNA miR-1298-5p into exosomes and excreting it out of cells.2.Glioma cells promote the malignant progression of glioma by retaining the pro-oncogenic miR-9-5p in the intracellular compartment.3.Glioma cells sort miR-1298-5p into exosomes and then exocytose it through the exocytosis protein hnRNPA2B1 binding to the UUCA sequence of miR-1298-5p.Part II Mechanism that tumor-suppressive miRNAs in glioma exosomes promotes the formation of immune suppressive microenvironmentObjectiveTo investigate the regulatory role of oncogenic miRNAs in glioma exosomes on the immune microenvironment in order to fully understand the mechanisms of miRNA sorting in glioma.Methods1.Oncogenic miRNA miR-1298-5p was transfected into MDSCs,and the transformation of immunosuppressive phenotype was detected by flow cytometry.2.The regulatory effect of miR-1298-5p on the secretion of cytokines by MDSCs was verified by PCR and Elisa assays.3.The miR-1298-5p-transfected MDSCs were co-cultured with CD8+T cells,and its effect on the proliferation ability of CD8+T cells was verified by flow detection technique.4.Prediction of miR-1298-5p target genes in MDSCs by starbase and other databases to verify its role in MDSCs.5.Multiple exosomal oncogenic miRNAs were overexpressed in THP1-induced macrophages to validate their ability to induce an immunosuppressive phenotype in macrophages.Results1.miR-1298-5p in glioma exosomes was able to promote the conversion of MDSCs to an immunosuppressive phenotype.2.miR-1298-5p could promote the secretion of NO and TGF-β by MDSCs,thus exerting the immunosuppressive ability of MDSCs.3.MDSCs transfected with miR-1298-5p inhibited the proliferation ability of CD8+T cells and weakened their killing effect on glioma cells.4.miR-1298-5p regulates the immunosuppressive ability of MDSCs by targeting MSH2 gene.5.Several exosomal oncogenic miRNAs have a role in promoting the immunosuppressive ability of immune cells.Conclusion1.miR-1298-5p in glioma exosomes can enhance the immunosuppressive ability of MDSCs.2.miR-1298-5p can target MSH2 in MDSCs to act.3.Multiple exosomal oncogenic miRNAs have the ability to modify the immune microenvironment of glioma.