The Role And Mechanism Of M~6A Methylation In Regulating CD8~+T Cell Senescence And Tumor Progression

BackgroundThe incidence of tumors increases rapidly with age.It has been reported that elderly patients have more tumor metastasis,worse overall survival,and responsiveness to therapies compared to younger patients.The immune system is a potent protective physiological system in mammals.Both innate and adaptive immunity of the immune system are susceptible to age-related changes,which are called immune senescence.Because of the importance of the immune system in controlling tumor progression,immune dysfunction may impair immune surveillance and thus promote tumor progression in the elderly.Although significant progression has been made in tumor immunology research over the past 20 years,detailed comparisons of the immune system effects on tumor progression have not been made in younger and elderly individuals.Our study suggests that CD8+T cells have a significant impact on tumor progression and elderly patient survival.Several studies have reported that T-cell numbers decline with age,because of a decrease in T-cell output due to thymic degeneration and atrophy.In this study,we analyzed the changes in CD8+T cells in young and aged individuals to further investigate how CD8+T cells in the immune system control tumor progression.m6A(N6-methyladenosine)is one of the most prevalent RNA modifications and abundant transcriptome modifications involved in regulating post-transcriptional and translational mechanisms.m6A modifications affect many biological processes,such as immune signaling and tumorigenic progression.m6A modifications can be deposited by a core methyltransferase complex consisting of METTL3 and METTL14 and modified by the demethylases ALKBH5 and FTO demethylation.As the core catalytic component of the methyltransferase complex,loss of METTL3 disrupts the processes of many physiological processes,such as hematopoiesis and memory formation.In addition,METTL3 functions as an oncogene in the development of many types of tumor and inhibits tumor cell proliferation by regulating the m6A levels of its downstream target RNAs.The fate of m6A-modified RNAs is then determined by m6A reading proteins(e.g.,YTH family proteins,etc.)that affect their splicing,translocation,stability,and translation.Overall,m6A modifications play a complex network regulatory role in fundamental cell biology processes.However,the role of m6A modifications in cellular senescence remains unknown.Therefore,based on the above considerations,this project focuses on 4 points:(1)the differences in tumor progression and prognosis between young and elderly individuals;(2)the role of CD8+ T cells in regulating tumor progression in elderly;(3)the relevance of altered m6A modifications in functions of CD8+ T cells;(4)the analysis of related phenomena and mechanisms of action using animal models,cellular models and clinical specimens.Part 1.Effect of age on tumor metastasis and prognosisObjectiveTo investigate the changes in tumor progression of different tumor models in young and aged mice,and the effect of age on tumor progression and prognosis in clinical patients.Methods(1)A subcutaneous transplantation tumor model using B16F10,4T1 and LLC to explore the tumor growth at different weeks of age,and the survival of tumor-bearing mice at different ages was analyzed by comparing the lung metastasis at different weeks of age with through imaging systems.(2)Lung metastasis and survival of young and aged mice were verified by establishing B16F10,4T1 tail vein and 4T1 in situ tumorigenic post-excision lung metastasis models.(3)To analyze the effect of age on tumor progression and its survival by using clinical patient’s data with different tumor types and following patient survival.(4)To analyze the effect of age on tumor progression and its survival using clinical patient’s data with different tumor types from TCGA databases.Results(1)Through different types of subcutaneous transplantation tumor mouse models,tumor proliferation was found to be faster in younger mice relative to aged mice.(2)Different types of tumor metastasis models revealed that tumors were more likely to develop lung metastasis and had poorer survival in aged mice.(3)By analyzing the information of patient’s data with different types,it was found that the prognosis of elderly patients was worse.(4)Analysis of the clinical information of patients with different tumors types according to the TCGA databases revealed that patients with metastases had a worse prognosis,and the prognosis of elderly patients was worse.ConclusionIn different tumor models,aged mice had more lung metastases and worse survival.In the clinical patient’s data of different types,the prognosis of elderly patients was worse.Age is a key factor influencing tumor progression and affects the prognosis of patients.Part 2.Effect of age on m6A methylation modifications in CD8+T cellsObjectiveTo investigate the difference in m6A modification of CD8+ T cells in young and elderly individuals and the consequent effect on CD8+T cells function.Methods(1)Using flow cytometry,we analyzed the differences in CD8+T cells in young and aged mice.And the correlation between CD8+T cells and patient survival was analyzed by TCGA database.(2)To construct a tumor metastasis model by removing CD8+T cells and to explore the effect of CD8+T cells on metastasis and survival.(3)To analyze the differences in the senescence status and function of CD8+T cells in young and aged mice using flow cytometry.(4)To analyze the difference in the overall m6A methylation level of CD8+T cells in young and aged mice by the m6A overall methylation assay kit.(5)To find key pathway affecting CD8+ T cells that undergo m6A methylation in CD8+T cells of young and aged mice by sequencing analysis of MeRIP-Seq and mRNA-Seq.Results(1)By comparing the CD8+T cells in young and aged mice,we found that the proportion of CD8+T cells was lower in aged mice.The age of patients was found to be negatively correlated with the infiltration of CD8+T cells.(2)Analysis of the TCGA database showed that the infiltration of CD8+ T cells was significantly negatively correlated with the metastasis and prognosis of patients.(3)It was found that after the removal of CD8+T cells,the tumor metastasis increased,and the survival became worse.(4)CD8+T cells in aged mice were in a state of senescence,and their proliferation was slowed down,and their function was reduced.(5)The overall m6A methylation level was reduced in CD8+ T cells of aged mice.(6)The sequencing of MeRIP-Seq and mRNA-Seq of activated CD8+T cells from young and aged mice revealed that the level of m6A modification was reduced in aged mice,and GO analysis revealed that the pathways of lymphocyte proliferation and activation were enriched in the set of m6A down-regulated and mRNA expression down-regulated genes.ConclusionIn aged mice,CD8+T cells are in a senescent state and have reduced function,which affects tumor progression and survival in aged mice.The age of the patient affects the infiltration of CD8+ T cells.The overall m6A methylation level is reduced in CD8+T cells of aged mice.Part 3.The m6A methyltransferase METTL3 affects CD8+T cell senescence via SykObjectiveTo investigate METTL3 as a key m6A methyltransferase that regulates Syk expression,which in turn affects the function of CD8+T cells.Methods(1)To identify key genes regulating CD8+T cell function by KEGG analysis of MeRIP-Seq and mRNA-Seq sequencing results.(2)To detect the difference of Syk expression in CD8+T cells of young and aged mice using RT-PCR and other experimental methods.(3)To detect the effect of Syk on CD8+T cell function by flow cytometry using inhibitors of Syk.(4)The expression of various methyltransferases and demethyltransferases on CD8+T cells in young and aged mice were examined by RT-PCR and Western Blotting.The results were validated by mRNA-Seq sequencing of CD8+T cells.(5)To analyze the relationship between METTL3 and Syk expression and targeting by RT-PCR,Western Blotting and to verify the correlation between METTL3 and Syk expression in clinical patients using GEPIA databases.(6)To validate the expression of METTL3 on senescent CD8+T cells using H2O2induced senescence model,and to further investigate the expression of Syk and downstream pathways in H2O2-induced senescence model by RT-PCR and Western Blotting.(7)To establish a mouse model of METTL3 knockdown on CD8+T cells and analyze the correlation between METTL3 knockdown and tumor metastasis and survival,as well as the effect on the expression of downstream target gene Syk.(8)To further analyze the correlation between METTL3 and Syk expression by screening METTL3 inhibitors and using RT-PCR and other experimental techniques.Results(1)Sequencing analysis by MeRIP-Seq and mRNA-Seq of CD8+T cells revealed reduced expression of Syk and decreased levels of m6A modification in aged mice.(2)In vitro experiments further verified the reduced expression of Syk in CD8+T cells from aged mice.(3)After the addition of Syk inhibitor,the expression of Syk was reduced and the function of CD8+T cells was decreased.(4)METTL3 expression was decreased in CD8+ T cells from aged mice.METTL3 expression was also reduced in the H2O2-induced senescence model.This was further validated by sequencing of CD8+T cell mRNA-Seq.(5)The expression of Syk was reduced after knockout METTL3.(6)The expression of both METTL3 and Syk was reduced in the H2O2-induced senescence model.(7)The expression of Syk was reduced after the addition of METTL3 inhibitor.(8)Detection of METTL3 regulation of CD8+ T cell function through Syk-PI3KAKT pathway using flow cytometry.ConclusionIn CD8+T cells of aged mice,METTL3 expression was decreased compared to younger mice,and it inhibited PI3K-AKT pathway activation through downregulation of Syk target genes,which in turn decreased CD8+ T cell function,leading to tumor progression in aged mice and affecting survival in aged mice.Part 4.Clinical significance of CD8+T cell Syk expression in young and elderly tumor patientsObjectiveTo investigate the impact of CD8+T cells Syk expression on the prognosis of clinical patients.Methods(1)To analyze the prognostic impact of METTL3 expression on CD8+T cells in clinical tumor patients using TCGA databases,and to analyze the correlation between METTL3 and CD8+T cell senescence and functional molecular expression.(2)To analyze the correlation between METTL3 and Syk expression using immunohistochemistry,and to further validate the prognostic impact of METTL3 and Syk co-expression on tumor patients using GEPIA database.(3)To analyze the effect of age on the expression of Syk in different tumor types by immunohistochemistry.The TCGA database was used to analyze the effect of Syk expression on the prognosis of patients.(4)To analyze the correlation between CD 8A and Syk expression using TCGA database,and to analyze the effect of co-expression of CD8A and Syk on the prognosis of patients.(5)To analyze the effect of age on CD8A expression in patients with different types of tumors using immunohistochemistry.(6)To analyze the correlation between age and CD8A expression and the effect of CD8A expression on the patient’s prognosis using TCGA database.Results(1)Patients with low expression of METTL3 on CD8+T cells have poorer survival.And METTL3 was correlated with CD8+T cell senescence and functional molecule expression.(2)METTL3 was significantly correlated with Syk expression,and the co-low expression of METTL3 and Syk was associated with poor prognosis of patients.(3)The expression of Syk was low in the tissues of aged patients,and the prognosis of patients in the Syk low expression group was found to be poorer using GEPIA database.(4)The expression of CD8A and Syk showed a positive correlation,and the colow expression of CD8A and Syk was associated with poor prognosis of patients.(5)In elderly patients,the expression of CD8A was relatively low.(6)The analysis of TCGA database showed that the expression of CD8A was negatively correlated with age.Moreover,low CD8A expression was associated with poor prognosis of patients.ConclusionIn elderly patients of tumor,CD8+T cells and the expression of METTL3 and Syk affect the survival of patients.METTL3 and Syk may be used as therapeutic targets for elderly patients of tumor.