Screening And Study The Mechanism Of Pathogenic Markers In Multiple Myeloma

Multiple Myeloma（MM）is a common hematologic malignancy caused by malignant clonal proliferation of plasma cells.Currently,MM is difficult to cure and seriously threatens people’s life and health,and it is easy to be misdiagnosed or underdiagnosed,resulting in delayed condition.Complex gene expression changes and chromosomal instability,as well as interactions between tumor cells and the bone marrow immune microenvironment,may collectively promote MM disease process and drug resistance.Therefore,understanding the biological characteristics of MM tumor cells,elucidating the biological mechanisms associated with MM occurrence and development,and discovering effective biomarkers and potential drug targets are conducive to the precise diagnosis and treatment of MM.In this dissertation,we firstly used single-cell transcriptome sequencing（ScRNA-seq）technology to study the oncogenes and potential immunotherapeutic targets of MM from the transcriptome perspective.Next,we used next-generation sequencing（NGS）technology to analyze the gene mutation spectrum of MM from the genome perspective.Finally,we analyzed the clinical immunological indexes and constructed the prediction model of the risk of acquiring MM disease,so as to provide references for diagnosis and treatment of MM.The main research contents are as follows:1.ScRNA-seq-based studies of MM markers and potential immunotherapeutic targets.Fewer studies have focused on the behavioral changes in the total population of cells within the MM bone marrow and explored their role in MM development.In this part,ScRNA-seq analysis and vitro experimental were performed on patients with different Revised International Staging System（R-ISS）staging of MM.This study identified a NKG7⁺cytotoxic plasma cell（PC）cluster that was significantly enriched in MM-II patients.The malignant PC cluster had significantly elevated MKI67 and PCNA expression,which was associated with poor prognosis and Epstein-Barr virus infection.In this part,we identified and validated that ribonucleic acid reductase regulatory subunit M2（RRM2）promoted MM cell proliferation,suggesting that RRM2 might serve as an pathogenic marker for MM.The percentages of CD8⁺T cells and NK/T cells decreased with R-ISS staging,suggesting plasticity of the MM immune microenvironment.CD8⁺T cells and NK/T cells in communication with myeloid cells and PCs.Several potential immunotherapeutic targets were identified,such as SIRPA-CD47 and MIF-CD74.These results suggested that RRM2 might serve as a pathogenic marker for MM.In addition,more malignant cell clusters might play a more important role in advancing MM.Bone marrow immune microenvironment might be a key factor influencing the evolution of MM cell clusters.2.NGS-based study of bone marrow PC gene mutation profiles in MM.Alterations in cell behavior are traced to changes in gene and protein expression.In order to explore the underlying genetic factors for the altered PC population,targeted exome sequencing of bone marrow PCs from 50 MM patients was performed using NGS technology to analyze their gene mutations.A total of 337 mutations were identified in MM,with mutation types including SNP,INS,and DEL.The most predominant mutation type was SNP.Analysis of the GSE6477 dataset,a total of 660 down-regulated genes and 587 up-regulated genes were identified.There were 33 genes common to the MM mutated genes and differentially expressed genes（DEGs）.Some of the mutated genes were closely associated with clinical immune indicators,such as IL-10 andβ₂M.Further analysis showed that the functions and pathways of these mutated genes were enriched in myeloid differentiation and hematopoietic regulation.In addition,we found that low expression of BCL6,BIRC3,HLA-DQA1 and VCAN was associated with poor prognosis in MM.The expression of BCL6,BIRC3 and HLA-DQA1 was found to be negatively correlated with the infiltration of myeloid regulatory T-cells.The expression of BCL,VCAN and HLA-DQA1 was positively correlated with the level of neutrophils.Markers screened by ScRNA-seq and NGS were found to be associated with RRM2 and VCAN possibly through TP53 by gene interaction analysis.RRM2 and BCL6 might be linked through GLRX.The above results suggested that mutations and low expression of BCL6,BIRC3,HLA-DQA1 and VCAN were associated with prognosis and immune cell infiltration in MM.The markers screened by ScRNA-seq and NGS might collectively affect the immune status of MM.3.Analysis of clinical immunological indicators of MM and construction of predictive model for the risk of acquiring MM disease.The ScRNA-seq and NGS suggested that the development of MM was associated with the immune microenvironment of patients.Therefore,a comprehensive analysis of the available clinical immunological indicators of MM might reveal patterns of immune behavior specific to MM patients.In this part,clinical immune indicators of MM were collected and studied with other patients with malignant plasma cell disease and B cell lymphoma（BCL）.The analysis revealed that the positivity of M protein was 94.1%and 74.9%in MM and BCL,respectively.FLCκand FLCλlevels were seen to be abnormal in all disease groups,but the highest overall levels were found in MM.The highest ratio of FLCκ/FLCλabnormalities were observed in MM.IL-10 andβ₂M were significantly different between these diseases,which may be related to the gene mutations identified by NGS analysis.Flow cytometry analysis showed significant differences in bone marrow B-cell counts between different diseases,with significantly lower levels in MM than in monoclonal globulinopathies of undetermined significance（MGUS）and BCL.The median CD4⁺T versus CD8⁺T and CD4⁺T/CD8⁺T ratios were reduced between MGUS,MM,and BCL,which suggested that abnormalities in the distribution of T-cell subsets were present in all of these disorders.That was consistent with the T-cell related conclusions from the ScRNA-seq analysis.It was suggested that the immune function status of MM is somewhat different from that of other malignant plasma cell diseases and BCL.It could be seen that this is subject to further analysis of the reasons for the differences in the relevant immune indicators.In this part,a single center risk prediction model for acquiring MM was established based on IL-10,β₂M and other clinical immunity indexes,and the performance of the prediction model was good.This study laid a certain foundation for the diagnosis and treatment of MM.In summary,we screened the pathogenic markers of MM using ScRNA-seq and NGS technology,and explored the possible mechanisms of these markers in MM by clinical samples and in vitro tests.Also,we found that the alteration of the immune microenvironment may be closely related to the development of MM,and further analysis of the clinical immune indexes suggested that the immune function status of MM is somewhat different from other malignant plasma cell diseases.The performance of the single center predictive model for acquiring MM risk based on immune indexes is good,which provides a basis for the diagnosis and treatment of MM.The results of this study may provide new ideas for further diagnosis and prognosis of MM.