RNA M~6A Modification Regulates The Differentiation And Immunosuppressive Function Of MDSC

ObjectivesThe concept of using the immune system to control tumor growth dates back to 1893,when William Coley used living bacteria as an immunostimulant to treat cancer[1].In the past few decades,tremendous progress has been made in understanding how cancer evades surveillance by the immune system,and studies have found that a variety of immune cells play an important role in this process,which in turn provides new ideas to overcome cancer immune escape[2].Bone marrow-derived suppressor cells(MDSCs)are an important part regulating immune responses during inflammation and cancer.MDSC is a group of heterogeneous cell populations derived from bone marrow stem/progenitor cells,mainly including immature granulocytes,dendritic cells,macrophages,and early undifferentiated myeloid precursor cells[3].In the case of tumors and chronic inflammation,the body will produce a variety of cytokines such as GM-CSF,G-CSF,VEGF and IL6.In the medullary cavity,these cytokines can induce abnormal differentiation of bone marrow progenitor cells,leading to the development of MDSCs[4].In humans,MDSCs are defined as a group of cells that co-express CD11b and CD33 but lack HLA-DR.They mainly include CD15+PMN-MDSC(polymorphic nuclear MDSCs)and CD14+ M-MDSC(mononuclear MDSCs).In mice,MDSCs are defined as a group of cells of CD11b+Gr1+,mainly including PMN-MDSCs of CD11b+Ly6G+ Ly6Clo and M-MDSCs of CD11b+Ly6G-Ly6Chi[5].MDSC expresses immunosuppressive genes such as ARG-1,NOS2,and IL-10,and produces NO and ROS to form immunosuppressive tumor microenvironment.It can also change its own lipid metabolism through genes such as OLR1,thereby suppressing T cell immunity and lead to immunosuppression[6].N6-methyladenosine(m6A)mRNA methylation modification is the most abundant mode of epigenetic modification in eukaryotic cells.It plays an important role in the normal development of mammals and the development of disease.It also contributes to the post-transcriptional modification of nuclear messenger RNA(mRNA),microRNA(miRNA),and long non-coding RNA(lncRNA)and is considered to be one of the most common,reversible and abundant internal modifications on RNA molecules[7].In mammals,the methyltransferase complex consisting of the enzyme subunit METTL3,the substrate recognition subunit METTL14 and the regulatory subunit WTAP catalyzes the target RNA in RRACH(where R=G or A,H=A,C or U)domain and adds a methyl group to the N6 site of adenine in the consensus sequence,which is called m6A.At the same time,mRNAs that have undergone m6A methylation modification can be cleared by methylases FTO and ALKBH5[8,9].At the molecular level,transcripts that are dynamically labeled by m6A can be read by methyl "reader" proteins to modify information on them,thereby altering mRNA metabolism in different ways.m6A modification affects almost every step of RNA metabolism,including mRNA processing,nuclear export,translation,degradation,and the formation and degradation of long non-coding RNA(lncRNA)and microRNA(miRNA),which are involved in all processes of biological development[10].Recently,more and more studies have found that m6A is closely related to the occurrence and development of cancer.m6A modification can participate in the self-renewal of tumor stem cells,promote the proliferation of cancer cells,and resistance to radiation therapy or chemotherapy[11].At present,m6A-modified related target genes have been found in a variety of cancer species,and the downstream related pathways involved in this process have been identified,which indicates that m6A-related enzymes and their modified sites may be new targets for cancer treatment;some studies have shown that Changes in the overall m6A methylation level of cells are related to the development of cancer and may be related to changes in various immune cells[8].Changes in the m6A methylation level of cells can be used as an indicator to identify the different stages of cancer and different subtypes of immune cells.Previous studies have shown that a variety of cytokines,transcription factors,signaling pathways,and epigenetic modifications are involved in the formation,polarization,and immunosuppressive function of MDSC[12].m6A methylation modification in the bone marrow Cell proliferation and differentiation play an important regulatory role[13],but the effects of m6A mRNA methylation modification and related methylases on MDSC have not been specifically reported.Therefore,this project aims to explore the regulatory role of changes in the overall m6A methylation level of cells on MDSC formation,polarization,and immunosuppressive functions,as well as methylases related to the formation of MDSCs,and to explore the formation and development of MDSCs.The new mechanism provides new research ideas for immunotherapy of cancer.MethodsBone marrow cells(BMC)were obtained from the femur of C57BL/6 mice and cultured in 1640 medium supplemented with GM-CSF and G-CSF for in vitro induction of MDSCs.CD11b+Gr+cells was detected by flow cytometry after 72 hours cells was compared with the original BMC,and the expression of genes related to the immunosuppressive function of MDSC was detected by RT-PCR,and the cell ratio of Ly6G+Ly6Clo and Ly6G-Ly6Chi were detected in CD11b+cells.Then,the m6A level of the induced cells was detected by the m6A methylation level detection kit.Next,while inducing MDSC,the global cell m6A methylation level inhibitor DAA was added,and the induction ratio was detected by flow cytometry and the proportion change of the two subpopulations was detected,and the expression change of immunosuppressive genes was detected by RT-PCR technology.Then we detected the m6A methyl modification level.By grinding the spleen of mice and obtain CD8+T cells by immunomagnetic bead cell sorting.We then activate these CD8+T cells with CD3/CD28 activation microbeads.After 72 hours,we incubate them with MDSCs induced by different methods.After 72 h of co-incubation,we detected the Ki67 expression of T cells and used the CFSE cell proliferation ability test to value the proliferation ability of T cells.RT-PCR was used to detect the changes of m6A-related enzymes in MDSC.It was found that the expression of METTL3 was significantly reduced,which may be related to the decrease of MDSC m6A methyl levels and the enhancement of immunosuppressive function.By comparison with the database,it was found that the prognosis of LUAD patients with low METTL3 expression was poorer.We then used magnetic beads to sort the peripheral blood MDSCs of LUAD patients and compared them with normal human peripheral blood mononuclear cells,and RT-PCR was used to verify that METTL3 expression was significantly reduced.Results1.The results of flow cytometry showed that the use of GM-CSF+G-CSF significantly increased the proportion of CD11b+Gr-1+cells,and the proportion of Ly6G+Ly6Clo cells in this group was higher.2.RT-PCR verified that the expression levels of ARG-1,NOS2,and TGF-? in MDSCs induced by GM-CSF+ G-CSF were significantly increased.3.Quantitative detection of m6A methylation showed that the level of m6A methylation modification in MD SC was significantly reduced.4.Quantitative detection of m6A methylation experiments showed that the use of DAA significantly reduced the level of m6A methyl modification in MDSC.5.Flow cytometry results showed that the use of DAA did not change the proportion of cells that induced MDSC,but the proportion of Ly6G+Ly6Clo cells decreased,and the proportion of Ly6G-Ly6Chi cells increased.6.RT-PCR verified that the expression of ARG-1,IL-10,OLR1,and TGF-? in MDSCs induced by GM-CSF+G-CSF+DAA significantly increased.7.Ki67 flow cytometry results and CFSE experimental results show that the application of DAA has enhanced the ability of MDSC to inhibit the proliferation of CD8+ T cells.8.The results of RT-PCR and Western blot experiments verified that the METTL3 expression in MDSC was significantly reduced,and the METTL3 protein level was further reduced after DAA was added.9.TCGA database verified that METTL3 expression in tumor tissues of patients with LUAD decreased,and it was related to the shortened OS.RT-PCR results showed that the expression of METTL3 in peripheral blood MDSCs of patients with LUAD was significantly reduced compared with normal people.ConclusionCompared with normal BMC,MDSC has a lower m6A methylation modification level,and the lower m6A methylation level changes the polarization direction of MDSC,which is related to the enhancement of its immunosuppressive function.The decrease of m6A methylation level in MDSC is related to the decrease of METTL3 expression,and the low METTL3 expression in the tumor tissue and peripheral blood MDSC of LUAD patients is related to its poor prognosis.