Study On The Role And Mechanism Of RNA-Binding Protein DAZAP1 In The Proliferation Of Multiple Myeloma

Background and ObjectiveMultiple Myeloma（MM）is the second most common hematological malignancy after nonHodgkin’s lymphoma.MM occurs during the maturation process of B cells,and the disease ofen progresses through the intermediate stages of monoclonal gammopathy of undetermined significance（MGUS）and smoldering multiple myeloma（SMM）,which then develops into an organic disease of terminal organ lesions.It is characterized by an abnormal increase of plasma cells,which secrete large amounts of monoclonal immunoglobulins,thereby causing bone destruction and immunodeficiency.The clinical manifestations include anemia,hypercalcemia,osteolytic bone disease and renal insufficiency.Currently,the main treatments on MM include radiotherapy,chemotherapy,hematopoietic stem cell transplantation（SCT）,etc.New drugs are used in clinic,such as proteasome inhibitors,immunomodulators and so on,which can effectively alleviate the condition of MM and prolong progression-free survival and overall survival of MM patients.Although there have been breakthroughs in the treatment of MM patients,recurrence and drug resistance are still complicated problems that hinder the cure of MM.Therefore,it is particularly important to further study the molecular pathological mechanism of MM and discover new biological targets for the prevention and treatment of MM.In this project,Azoospermia-Associated Protein 1（DAZAP1）was screened out based on the gene library,and it was differentially expressed between MM patients and normal people.DAZAP1 was overexpressed in MM cells to conduct phenotypic Studies,such as cell proliferation,cell cycle in vitro,and validate by constructing a xenograft tumor model in NOD/SCID mice in vivo.We performed variable splicing analysis though RIP-seq to find the downstream genes of DAZAP1.All experiments are aim to further investigate the molecular mechanism of DAZAP1,and to provide new targets and experimental basis for the diagnosis and treatment of MM.Methods1.The MM clinical database was used to analyze the relationship between the expression of DAZAP1 and the survival of MM patients.2.Lentivirus transfection system was used to construct DAZAP1 Empty Vector（EV）and DAZAP1 Overexpression（OE）MM cell lines,and the transfection efficiency was verified by WB（Western blot）assay;The CCK8 assay was used to detect the effect of overexpression of D AZAP1 on the proliferation of MM cells;The agar colony formation assay was used to detect the effect of overexpression of DAZAP1 on the proliferation of MM cells;and flow cytometry was used to detect the influence of overexpression of DAZAP1 on the mitotic cycle of MM cells.The NOD/SCID immunodeficient mice xenograft tumor model was constructed to verify the impact of overexpression of DAZAP1 on MM cell growth in vivo.3.After performimg RNA immunoprecipitation（RIP）experiments,high-throughput sequencing andusing rMARTs software to perform alternative splicing analysis to find possible downstream targets of DAZAP1.Based on database comparison and combining with MM patient gene library,the downstream targets of DAZAP 1 were further screened out.The splicing effect of target was verified by RIP-qPCR and PCR experiments.4.The signal pathway involved in DAZAP1 regulation was analyzed according to RIP-seq.Transient transfection of two different transcripts of KITLG,spliced before and after in HEK293 and MM cells,were used to verify the transient efficiency by WB and PCR assay.To explore the effect of DAZAP1 on the splicing of KITLG on related signaling pathways.Results1.Based on the analysis of MM patient datebase,the differentially expressed gene DAZAP1 was screened out.It was found that the expression of DAZAP1 was closely related to the disease progression of MM patients,and was significantly correlated with the survival rate of MM patients.The survival rate of MM patients with low expression of DAZAP1 was significantly higher than that of MM patients with high expression of DAZAP1.2.WB results proved that CAG EV,CAG DAZAP1-OE and OCI EV,OCI DAZAP1-OE MM stable transfected MM cell lines were successfully constructed.CCK8 assay showed that overexpression of DAZAP1 significantly promoted the proliferation of MM cells,while knockdown of DAZAP1 significantly inhibited the proliferation rate of MM cells.Flow cytometry results showed that the effect of DAZAPI on the proliferation of MM cells was not caused by regulating the cell cycle distribution.The MMxenograft tumor model displayed that the weight and volume of subcutaneous tumors after overexpression of DAZAP1 were absolutely larger than those of the control group.3.Based upon RIP high-throughput sequencing combined with alternative splicing analysis,the findings showed that overexpression of DAZAP1 could lead to alternative splicing of multiple genes.The downstream gene KITLG was obtained through database comparison and survival curve analysis.The sixth exon of KITLG gene could be skipped,which resulting in two different transcripts:transcript KITLG（NM₀03899.5）and transcript KITLG（NM₀00994.6）.RIP-qPCR and PCR experiments showed that DAZAP1 had a splicing effect on KITLG mRNA.The expression of transcript KITLG（NM₀00899.5）was decreased while KITLG（NM₀03994.6）was increased after overexpression of DAZAP1.Two transcriptional plasmids of KITLG were instantaneously transferred in HEK293 and MM cells.WB showed that KITLG spliced by DAZAP1 enhanced the phosphorylation level of ERK and regulated MAPK signaling pathway.4.Further analysis of RIP-seq,it is found that the role of DAZAP1 in MM was closely related to the MAPK signaling pathway through GO and KEGG enrichment analysis.RAS activity assay demonstrated that overexpression of DAZAP1 increased RAS activity,which in turn caused the elevated phosphorylation level of ERK,as result of promoting MM cell proliferation to a certain extent.ConclusionIn this study,we explore the molecular biological function of DAZAP1 in MM and demonstrate its splicing effect on downstream target gene KITLG via using high-throughput sequencing and modern molecular biology methods.It is also proved that DAZAP1 promotes MM cell proliferation by regulating MAPK signaling pathway.This study is intended to reveal the molecular mechanism of DAZAP1 promoting the proliferation of MM cells from at epigenetic level,to provide a new molecular target for the targeted therapy of MM,and to provide an experimental basis and theoretical basis for clinically conquering MM.