| Background and objective:Multiple myeloma(MM)is a malignant disease of the hematological system characterized by abnormal proliferation of clonal plasma cells and excessive production of monoclonal immunoglobulins.Patients with MM often have signs and symptoms such as bone pain,anemia,renal impairment,and infection.Although the advent of novel drugs and improved treatment strategies have led to a better prognosis for MM patients,early identification,relapse refractoriness and drug resistance for MM patients remains challenging.Current studies suggest that multiple factors are involved in the development of MM,including genetic abnormalities,alterations in the bone marrow microenvironment and epigenetic abnormalities.As an important component of epigenetics,N6-methyladenine(m6A),plays an important role in the development and progression of many neoplastic diseases,including MM.Therefore,studying the biological roles of m6 Arelated genes in MM and further clarifying their pathogenic mechanisms and pathogenesis can contribute new ideas for optimizing the diagnosis and treatment of MM and developing target therapys.This research aims to investigate the expression of the key m6 A modification enzyme RBM15 in MM patients and its correlation with clinical indicators and diagnostic value,and further investigate the molecular mechanism of RBM15 regulating MM cell autophagy through m6 A methylation modification,and to clarify its role in the development of MM,so as to provide theoretical basis for identifying new clinical treatment targets for MM.Part Ⅰ: Screening and identification of m6A key genes closely related to multiple myeloma by bioinformatic methodMethods:(1)Bone marrow from 15 patients newly diagnosed with MM at Shanghai Changzheng Hospital and 15 normal donors at Shanghai First People’s Hospital were collected between October 2021 and October 2022,from which CD138-positive(CD138+)plasma cells were sorted,and the overall m6 A modification levels of both groups were detected by m6 A RNA methylation quantification kit(colorimetric method);(2)The overall m6 A modification levels of several common myeloma cell lines,including KM3,U266,LP1,RPMI8226,and NCI-H929,were examined by using m6 A RNA methylation quantification kit(colorimetric method);(3)Microarray datasets of healthy donors and MM were downloaded from the Gene Expression Omnibus(GEO)and preprocessed and combined with differential gene expression analysis and survival analysis to screen for m6A-relatede factors;(4)We randomly collected peripheral blood from 22 healthy examinations and 22 MM patients at Shanghai Changzheng Hospital between October 2021 and December 2021,and q RT-PCR was performed to detect the expression of RBM15 in bone marrow CD138+ cells and peripheral blood mononuclear cells(PBMCs)in the control group and MM patients.Results:(1)MM patients have significantly higher m6 A modification level than healthy controls,whereas MM cell lines,including KM3,U266,LP1,RPMI8226 and NCIH929,have significantly higher m6 A modification level than normal plasma cells;(2)The results of bioinformatic analysis suggest that RBM15 is associated with the development and prognosis of MM;(3)RBM15 is aberrantly highly expressed in both bone marrow CD138+ cells and peripheral blood PBMCs of MM patients.Conclusions: RBM15 is highly expressed in MM patients and is associated with poor prognosis in MM,and is expected to serve as a biomarker for MM treatment and prognosis.Part Ⅱ: Correlation of RBM15 expression levels in PBMCs of patients with multiple myeloma with clinical indicatorsMethods:(1)Fifty newly diagnosed patients,20 patients who were evaluated as disease progression and 30 patients with complete remission of MM and 45 healthy controls were collected at Shanghai Changzheng Hospital between January 2022 and November 2022,and the expression of RBM15 in PBMCs of MM patients was detected by q RT-PCR;(2)The correlation between the age,gender,type,hemoglobin,albumin,serum creatinine,β2 microglobulin,serum calcium,lactate dehydrogenase,DS stage,ISS stage,R-ISS stage and their RBM15 expression levels in PBMCs was analyzed in the above newly diagnosed MM patients,and the value of RBM15 in the diagnosis of MM was explored with the receiver operating characteristic curve(ROC).Results:(1)The expression level of RBM15 was significantly higher in both newly diagnosed MM patients and patients with progressive disease than in patients with complete remission MM and healthy physical examiners;(2)The expression level of RBM15 was significantly higher in MM patients with ISS stage III than in those with stage I/II,was positively correlated with serum β2 microglobulin and blood creatinine levels; and in patients typed as Ig G/Ig A,their RBM15 expression was positively correlated with serum Ig G/Ig A levels;and the results of ROC analysis showed that the area under the curve(AUC)of RBM15 as an independent diagnostic index was 0.778,and the AUC was 0.995 after combining serum albumin and hemoglobin,and 0.818 after combining blood creatinine.Conclusions: The expression level of RBM15 correlates with disease severity in MM patients and has the potential to be a diagnostic biomarker for MM.Part Ⅲ: Biological role of RBM15 in multiple myeloma cellsMethods:(1)Exploring the potential mechanism of RBM15 in MM by performing Gene Set Enrichment Analysis(GSEA)in the MM dataset GSE24080;(2)The expression of RBM15 in MM cell lines was detected by q RT-PCR,and RBM15 was stably knocked down and overexpressed in MM cells,while the knockdown and overexpression efficiency was verified by q RT-PCR and Western blot(WB);(3)Detecting the overall m6 A modification levels in MM cells after knockdown or overexpression of RBM15;(4)Examining the effects of knockdown or overexpression of RBM15 on the proliferation,apoptosis and autophagy of LP1,RPMI8266 and U266 cells.Results:(1)GSEA results suggest that RBM15 may be involved in MM cell autophagy and apoptosis;(2)RBM15 was highly expressed in LP1,RPMI8226 and NCIH929 cells and lowly expressed in KM3 and U266 cells,whereas the constructed knockdown or overexpression lentiviral vector resulted in stable knockdown of RBM15 in LP1 and RPMI8226 cells and stable overexpression in U266 cells;(3)Knockdown of RBM15 significantly decreased the overall m6 A modification level in LP1 and RPMI8266 cells,while overexpression of RBM15 significantly increased the overall m6 A modification level in U266 cell;(4)overexpression of RBM15 promotes proliferation and autophagy in MM cells,while knockdown of RBM15 inhibits proliferation and autophagy and induces apoptosis in MM cells.Conclusions: RBM15 is mainly involved in the autophagy of MM cells.Downregulation of RBM15 inhibits MM cell proliferation and autophagy,while inducing apoptosis in MM cells.Part Ⅳ: Screening and identification of downstream target of RBM15Methods:(1)Screening downstream target genes of RBM15 by combined m6 A immunoprecipitation conjugate sequencing(Me RIP-seq),mRNA sequencing and autophagy-related gene set;(2)Validation of the correlation between RBM15 and HSPA8 expression by q RT-PCR and GEO database and EMBL-EBI database;(3)q RT-PCR and WB verifying the effect of RBM15 on HSPA8 mRNA and protein expression;(4)Examining the effect of RBM15 on the stability of HSPA8 by mRNA stability assay;(5)WB detects the rescue effect of knockdown or overexpression of HSPA8 on autophagy in MM cells after knockdown or overexpression of RBM15.Results:(1)Knockdown of RBM15 decreased the m6A methylation modification level of autophagy-related gene HSPA8 and increased its expression level;(2)The RBM15 expression was negatively correlated with HSPA8 expression;(3)RBM15 leads to decreased HSPA8 mRNA stability;(4)Knockdown or overexpression of RBM15 alone in MM cells inhibits or promotes MM cell autophagy,while simultaneous knockdown or overexpression of HSPA8 partially reverses the effect of knockdown or overexpression of RBM15 alone on autophagy in MM cells.Conclusions: RBM15 affects the stability and expression of the target gene HSPA8 mRNA by regulating its level of m6 A methylation modifications,which in turn regulates MM cell autophagy.SummaryIn this thesis,we firstly found that RBM15,a key m6A modifying enzyme,is closely related to the development and prognosis of MM through bioinformatic method,and further validated and explored its clinical potential value through clinical samples.In vitro cell experiments reveal that RBM15 affects the stability of target HSPA8 mRNA through m6 A methylation modification,which in turn regulates MM cell autophagy. |
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