Study On The Mechanism Of Multiple Myeloma Proliferation Induced By BUB1B Via Centrosome Isolation Pathway

Multiple Myeloma?MM?is a malignant plasma cell disease.Its tumor cells originate from plasma cells in the bone marrow.Plasma cells are cells in which B lymphocytes develop to the final functional stage.Multiple myeloma is often accompanied by multiple osteolytic lesions,hypercalcemia,anemia,and kidney damage.With the high incidence of multiple myeloma in recent years,a large number of new therapies for MM have emerged,including general treatment,hematopoietic stem cell transplantation,radiotherapy and chemotherapy.Chemotherapy includes?targeting drugs such as proteasome inhibitors?bortezomib,carfilzomib?and immunomodulators;glucocorticoids such as dexamethasone,prednisone,etc.?.Because malignant tumors have the characteristics of recurrence,metastasis,and drug resistance,multiple myeloma is still incurable at this stage.Therefore,research on multiple myeloma is imminent.In the early stage of this project,high-throughput screening revealed that the BUB1B gene?BUB1 Mitotic Checkpoint Serine/Threonine Kinase B?is closely related to the occurrence and development of MM patients.And through gene chip technology,MM patient clinical data and HOVON database,etc.,confirmed that BUB1B gene is closely related to the occurrence and development of MM patients.Therefore,the development of the subject proceeded.First,by constructing stable cell lines of BUB1BOE and BUB1BKD,the effects of BUB1B overexpression and knockdown on the activity and cycle of multiple myeloma cells were studied by MTT experiments and flow cytometry.Experimental results found that BUB1BOE/BUB1BKD+Dox?BUB1BKD after doxycycline induction?promoted/inhibited cell proliferation,and BUB1BOE/BUB1BKD+Dox prolonged/shortened the proportion of mitotic G2/M phase.The above results can only explain that BUB1B plays a role in MM cells in phenotype studies,and its specific mechanism is not clear.Based on the above research,based on the BUB1B gene is involved in the separation of cell cycle mitotic chromosomes,so through exon sequencing,Giemsa staining,immunofluorescence and other methods to detect the overexpression of BUB1B on chromosome instability?Chromosomal instability,CIN?and drug resistance.Through the Co-IP experiment to explore the correlation between BUB1B protein and upstream and downstream proteins.The experimental results found that exon sequencing,Giemsa staining,and immunofluorescence experiments showed that BUB 1BOE compared with BUB1BWT cell line,its DNA copy number loss,duplication and misallocation rate increased,cell karyotype changed,chromosomal instability increased.Under the intervention of bortezomib and doxorubicin,the resistance of BUB1BOE cell line was enhanced.Western blot and Co-IP experiments found that BUB IB protein expression was positively correlated with centrosome-related proteins CEP 250 and CEP 170.BUB1B protein interacted with centrosome protein CEP 170 and centrosome protein CEP 250.CIN is the main cause of tumor development,but the mutual regulation of the bone marrow microenvironment?circular RNA,exosomes,miRNAs,etc.?plays an important role in multiple myeloma.Based on the above-mentioned related research on BUB1B,and the research on circular RNA in recent years has been particularly popular,so this topic is to conduct research on BUB1B circular RNA.In the early stage of the research group,the circular RNA chip screen found that the BUB1B circular RNA expressed by normal human plasma cells was lower than that of patients with multiple myeloma.In order to further explore the role of BUB1B circular RNA in multiple myeloma.This subject explores the expression of BUB1B circular RNA?Has_circ₀034565,Has_circ₀034566,Has_circ₀034567?at the RNA level in multiple myeloma through RT-PCR,qRT-PCR and other experiments,and finds that only BUB1B circular RNA?Has_circ₀034566?has at the RNA level Expression,so BUB1B circular RNA?Has_circ₀034566?was selected as the research object.Electroporation experiments,Western blot,protein profiles,MTT experiments,Giemsa staining,and immunofluorescence experiments explored whether BUB1B circular RNA?Has_circ₀034566?was expressed at the protein level and phenotyped in MM cells.The experimental results show that BUB1B circular RNA?Has_circ₀034566?cell line has the ability of protein translation.MTT experiment,Giemsa staining and immunofluorescence experiments show that overexpression of BUB 1B circular RNA?Has_circ₀034566?will promote the proliferation of MM cell line,leading to the karyotype Change and enhance CIN.To sum up,this paper explores the mechanism of BUB1B gene acting on MM cells by exploring the role of BUB1B in multiple myeloma cells;At the same time,the mutual regulation of the bone marrow microenvironment?BUB1B circular RNA?of multiple myeloma is studied to explore the commonality of the linear BUB1B and BUB1B circular RNA acting on the MM mechanism,which will provide further research on how BUB1B specifically regulates MM cells basis.