| Background:Multiple myeloma(MM)is a plasma cell malignancy characterized by malignant proliferation of plasma cells in the bone marrow with monoclonal immunoglobulin secretion,which accounts for 10%of all hematologic malignancies and has a low five-year survival rate.The pathogenesis of MM is complex,and in the past half century,the introduction of new drugs,such as bortezomib,and the use of hematopoietic stem cell transplantation have transformed the rapidly lethal MM into a chronic and manageable disease,extending the survival of most patients.However,MM lacks symptoms in its early stages and current tests are difficult to achieve recognition of the disease’s onset.Circular RNAs(circ RNAs)are widespread and diverse endogenous non-coding RNAs found in eukaryotic cells.the high stability and specific expression pattern of circ RNAs allow them to serve as potential diagnostic and prognostic biomarkers and play an important role in tumors.Autophagy,the process of engulfing one’s own cytoplasmic proteins or organelles and encapsulating them into vesicles and fusing with lysosomes to form autophagic lysosomes that degrade their encapsulated contents,plays a crucial role in the pathophysiology of MM.With the development of sequencing technology and the increasing understanding of circ RNAs,more and more studies suggest that circ RNAs may play an important regulatory role in disease activity,enhancing or inhibiting tumor development by regulating the autophagic process of tumor cells.However,to date,no circ RNAs have been reported to directly regulate autophagy in the pathological process of MM.Purpose:The aim of this study is to screen and obtain novel molecular markers that can be used for MM diagnosis and prognosis assessment by circ RNA high-throughput sequencing technology and correlation analysis of clinical parameters;and then investigate the role of circ RNA in regulating MM cell autophagy and the molecular mechanism through the study of MM cell line biological function and autophagy mechanism.Part I:Screening and identification of hsa＿circ＿0042819 in multiple myelomaMethods:(1)High-throughput sequencing was applied to screen out circ RNAs aberrantly expressed in bone marrow CD138~+cells of MM patients,and the significantly upregulated circ RNAs were screened by pathway enrichment analysis and quantitative polymerase chain reaction(q PCR).(2)We detected the expression of circ RNA in peripheral blood samples from MM patients and MM cell lines by q PCR to further validate the expression of circ RNA in MM.(3)Specially designed circular divergent primers and linear convergent primers were designed for PCR experiments and agarose gel electrophoresis,and the circular structure of circ RNA was verified by Sanger sequencing,and the nucleoplasmic separation experiments and fluorescence in situ hybridization(FISH)was performed to confirm the localization of circ RNA in MM cells.Results:(1)We obtained the aberrantly highly expressed circ RNA:hsa＿circ＿0042819 by screening with high-throughput sequencing technology and pathway enrichment analysis.(2)hsa＿circ＿0042819 is significantly highly expressed in peripheral blood and MM cell lines of MM patients;(3)We confirmed that hsa＿circ＿0042819 is a circ RNA with a circular structure and is stably expressed in the MM cytoplasm.Part II:Correlation analysis of hsa＿circ＿0042819 with clinical test indicators in multiple myeloma patientsMethods:(1)We expanded the sample size to detect the expression of hsa＿circ＿0042819 in MM patients at different stages of disease(newly diagnosed,progressive and complete remission stages).(2)We collected clinical data on gender,age,disease staging,stage,albumin,hemoglobin,lactate dehydrogenase,β2-microglobulin,blood creatinine,and blood calcium in patients with newly diagnosed MM patients,and analyzed the correlation between hsa＿circ＿0042819expression and clinical indicators.(3)We explored the diagnostic efficacy of hsa＿circ＿0042819 in MM by receiver operating characteristic(ROC)curves.(4)We explored the relationship between hsa＿circ＿0042819 and MM prognosis by Kaplan-Meier survival analysis.Results:(1)Patients with high hsa＿circ＿0042819 expression have higher tumor load and more rapid disease progression.(2)hsa＿circ＿0042819 expression levels were positively correlated with lactate dehydrogenase,uric acid,serumβ2-microglobulin,blood calcium,and M protein,and negatively correlated with hemoglobin levels.hsa＿circ＿0042819 expression was significantly correlated with MM clinical indicators.(3)The results of ROC curve analysis showed that the area under the curve(AUC)of hsa＿circ＿0042819 as an independent diagnostic index was 0.714.The AUC could reach more than 0.95 after combining hemoglobin,albumin and immunoglobulin,which could achieve better diagnostic efficacy either singly or in combination.(4)Patients with high expression of hsa＿circ＿0042819 had significantly shorter progression-free survival(PFS)and poorer prognosis.Part III:Study on the biological function of hsa＿circ＿0042819 in multiple myeloma cellsMethods:(1)We explored the effects of knockdown of hsa＿circ＿0042819 on MM cell proliferation through in vitro and in vivo experiments.(2)We applied multiple cell death inhibitors to determine the specific triggers of hsa＿circ＿0042819-induced cell death.(3)We investigated the effect of hsa＿circ＿0042819 on MM cell autophagy to further clarify its role in MM.Results:(1)Knockdown of hsa＿circ＿0042819 reduced the proliferation of MM cells both in vitro and in vivo.(2)The addition of autophagy inhibitors and apoptosis inhibitors significantly reduced knockdown of hsa＿circ＿0042819-induced cell death.(3)Knockdown of hsa＿circ＿0042819 induces enhanced autophagy and autophagic cell death in MM cells.Part IV:Screening and identification of hsa＿circ＿0042819 target binding proteins that regulate autophagy in multiple myeloma cells Methods:(1)We screened and validated the RNA binding protein(RBP)that may bind to hsa＿circ＿0042819 by RNA pull down assay(RNA pull down)and RNA immunoprecipitation(RIP)assay.(2)MM cells stably infected with sh NC and sh-hsa＿circ＿0042819 were applied for m RNA sequencing.The downstream target genes were searched by database prediction,q PCR and validated by RIP experiments.(3)We investigated the effect of hsa＿circ＿0042819/RBP on MM cell proliferation and apoptosis through specific m RNA and explored the specific mechanism by which hsa＿circ＿0042819/RBP affects MM cell autophagy through regulation of m RNA.Results:(1)The target binding protein of hsa＿circ＿0042819:Up-frameshift1(UPF1)was obtained by screening.(2)The regulatory m RNA of hsa＿circ＿0042819/UPF1:Calcium/calmodulin dependent protein kinase kinase 2(CAMKK2)was obtained by screening.(3)hsa＿circ＿0042819 can regulate CAMKK2 m RNA through binding to UPF1,which in turn affects MM cell autophagy.knockdown of CAMKK2 significantly counteracts knockdown of hsa＿circ＿0042819-induced autophagic flux,microtubule-associated protein1 light chain 3 beta(MAP1LC3B)conversion and degradation of sequestosome 1(p62/SQSTM1).Conclusion:1.hsa＿circ＿0042819 is a circ RNA with a stable loop structure and localized in the MM cytoplasm,and is stably highly expressed in bone marrow and peripheral blood of MM patients and MM cell lines.2.hsa＿circ＿0042819 is abnormally upregulated in the peripheral blood of patients with primary and progressive MM and is closely associated with disease progression,with the potential to become a biomarker for clinical diagnosis and prognosis of MM.3.Stable knockdown of hsa＿circ＿0042819 induces enhanced autophagy and autophagic cell death in MM cells.4.hsa＿circ＿0042819 can regulate the expression of CAMKK2 through binding to UPF1,which in turn affects MM cell autophagy. |
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