Regulation Of TRIP13 On Proliferation And Apoptosis Of Chronic Lymphocytic Leukemia Cells And Its Mechanism

Background and AimsThe treatment of chronic lymphocytic leukemia(CLL)is gradually transformed from traditional chemotherapy to molecular targeted drug therapy.Therefore,the exploration of new therapeutic targets and their mechanisms of action have become the focus of current research.Thyroid hormone receptor interactor 13(TRIP 13)is a member of the AAA+ ATPase superfamily related to a variety of cellular activities,and it plays important roles in meiotic homologous recombination,silencing and activation of mitotic checkpoint,and repair of DNA double-strand breaks.Moreover,TRIP 13 has been proven to be highly expressed in a variety of human tumors and is involved in the occurrence and development of tumors.It is a promising new target for treatment and prognostic indicator,but there are few related studies in CLL.Therefore,this study aimed to explore the regulatory effect of TRIP 13 on the proliferation and apoptosis of CLL cells and its possible molecular mechanism by knocking down/overexpressing TRIP 13 of the cell lines Granta-519 and JVM-2.Methods1.Isolate CD19+B lymphocytes from peripheral blood samples of CLL patients and normal people,and use real-time fluorescent quantitative PCR to detect the expression of TRIP13 of normal B lymphocytes,B lymphocytes of CLL patients and the cell lines Granta-519 and JVM-2.2.Use lentivirus infection technology to knock down/overexpress TRIP 13 of Granta-519 cells and JVM-2 cell and construct their control cells.Use a fluorescence microscope to determine the efficiency of lentivirus infecting the cells.Use real-time quantitative PCR technology and western blot to evaluate the effect of knockdown/overexpression.3.Use CCK-8 method to detect the proliferation ability of Granta-519 cells and JVM-2 cells.Start from 72h after lentivirus infection and detect a total of five time points(24h,48h,72h,96h and 120h).4.Five days after lentiviral infection,use Annexin V-APC single staining method to detect the apoptotic rate of Granta-519 cells and JVM-2 cells.5.Five days after lentiviral infection,use PI staining method to detect the distribution of cells in each phase of the cell cycle of Granta-519 cells and JVM-2 cells.6.Use western blot to detect the expression of P53,MDM4 and Bcl-2 in Granta-519 cells and JVM-2 cells.Results1.Compared with normal B lymphocytes,TRIP 13 mRNA levels of B lymphocytes from CLL patients,Granta-519 cells and JVM-2 cells increased significantly.2.The efficiency of knocking down TRIP 13 in Granta-519 cells and JVM-2 cells using lentivirus was 53.8%and 80.3%,respectively;the efficiency of overexpressing TRIP 13 in Granta-519 cells and JVM-2 cells using lentivirus was 2.43 times and 2.64 times,respectively.3.Compared with control cells,the proliferation of Granta-519 cells and JVM-2 cells after knocking down TRIP 13 was significantly reduced,and the proliferation of Granta-519 cells and JVM-2 cells after overexpressing TRIP 13 was significantly enhanced.4.Compared with control cells,the apoptosis rate of Granta-519 cells and JVM-2 cells after knocking down TRIP 13 increased significantly,and the apoptosis rate of Granta-519 cells and JVM-2 cells after overexpressing TRIP 13 decreased significantly.5.Compared with control cells,knocking down TRIP 13 in Granta-519 cells promoted G0/G1 phase arrest,and knocking down TRIP 13 in JVM-2 cells promoted G0/G1 phase arrest and G2/M phases arrest.6.Compared with control cells,the expressions of P53 protein decreased in Granta-519 cells and JVM-2 cells after knocking down TRIP13,and the expression of P53 protein increased in JVM-2 cells after overexpressing TRIP13.7.Compared with control cells,the expressions of MDM4 protein increased in Granta-519 cells after knocking down TRIP 13,and the expressions of MDM4 protein decreased in Granta-519 cells and JVM-2 cells after overexpressing TRIP13.8.Compared with control cells,the expression of Bcl-2 protein decreased in Granta-519 cells after knocking down TRIP 13,and the expression of Bcl-2 protein increased in JVM-2 cells after overexpressing TRIP13.Conclusions1.The expression level of TRIP 13 in CLL cells is significantly higher than that in normal B lymphocytes.2.TRIP 13 promotes the proliferation of CLL cells and inhibits the apoptosis of CLL cells.3.TRIP 13 affects the proliferation and apoptosis of CLL cells by participating in the regulation of the cell cycle.4.TRIP 13 in CLL cells promotes the expression of Bcl-2 and P53 proteins and inhibits the expression of MDM4 protein.