Effect Of CPEB4 On The Biological Function Of Chronic Myeloid Leukemia Cells

Objective:Cytoplasmic polyadenylation element binding protein 4(CPEB4)is a member of the cytoplasmic polyadenylation element binding protein family(CPEBs),a sequence-specific RNA-binding protein that promotes m RNA translation,recombination of CPEB4 regulated gene expression is a general mechanism of tumor development.At present,there is no research report on CPEB4 in chronic myeloid leukemia at home and abroad.1.To investigate the expression of CPEB4 in K562 cells of chronic myeloid leukemia cells,by changing the expression of CPEB4 protein in K562,the effects of CPEB4 on proliferation,apoptosis,migration and cycle of K562 cells were investigated.2.To explore the molecular mechanism of CPEB4 regulating proliferation,apoptosis,migration and cycle of K562 cells.Methods:1.Chronic myeloid leukemia cell K562 was cultured in vitro.2.Detection of CPEB4 expression in normal leukocytes and K562 cells by Western blot.3.Culture Escherichia coli DH5?(plasmid vector)to amplify the plasmid;extract the plasmid;The four plasmids pcDNA3.1(+)-His-CPEB4,pPLK+Puro-CPEB4 shRNA,pcDNA3.1 and pPLK+Puro were transfected into K562 cells by electroporation.4.After electroporation of K562 cells,the expression of CPEB4 protein was detected by western blot.5.The proliferation level of cells in different treatment groups was detected by CCK-8 reagent,and OD450 was detected by a microplate reader.6.Flow cytometry was used to detect the apoptosis level and cell cycle of cells in7.different treatment groups.8.Transwell chamber was used to detect the migration level of K562 cells in different treatment groups,and the number of cell migration was observed under the microscope.9.Western blot was used to detect the protein molecules involved in the proliferation,apoptosis,migration and cycle of K562 cells.Results:1.CPEB4 expression results Compared with normal white blood cells,the expression of CPEB4 protein in K562 cells was higher(P<0.01).2.Transfection efficiency results Compared with K562 cells transfected into pcDNA3.1 plasmid(His-CPEB4 null),the expression of CPEB4 was increased in K562 cells transfected with pcDNA3.1(+)-His-CPEB4 plasmid(His-CPEB4)(P<0.01).The expression of CPEB4 was decreased in K562 cells transfected with pPLK+Puro-CPEB4 shRNA plasmid(shRNA-CPEB4)compared with K562 cells transfected with pPLK+Puro plasmid(shRNA-CPEB4 null)(P<0.001);Compared with normal K562,K562 cells transfected into pcDNA3.1 plasmid and pPLK+Puro plasmid had no statistically significant expression of CPEB4.3.The results of cell proliferation and molecular protein involved in regulation Compared to K562 cells transfected into pcDNA3.1 plasmid(His-CPEB4 null),K562 cells transfected into pcDNA3.1(+)-His-CPEB4 plasmid(His-CPEB4)showed slower proliferation(P<0.05),AKT(P<0.01),p-AKT(P<0.001)protein expression decreased;Compared to K562 cells transfected into pPLK+Puro plasmid(shRNA-CPEB4 null),K562 cells transfected into pPLK + Puro-CPEB4 shRNA plasmid(shRNA-CPEB4)showed faster proliferation(P < 0.001),AKT(P <0.01),p-AKT(P < 0.001)protein expression increased.4.The results of cell apoptosis and molecular protein involved in regulation ompared with K562 cells transfected into pcDNA3.1 plasmid(His-CPEB4 null),the number of apoptosis in K562 cells transfected into pcDNA3.1(+)-His-CPEB4 plasmid(His-CPEB4)was increased(P<0.01),the expression level of BCL-2(P<0.05)was decreased;compared with K562 cells transfected into pPLK + Puro plasmid(shRNA-CPEB4 null),the number of apoptosis in K562 cells transfected into pPLK +Puro-CPEB4 shRNA plasmid(shRNA-CPEB4)was decreased(P<0.05),the expression level of BCL-2(P<0.001)was increased.5.The results of cell cycle and molecular protein involved in regulation Compared with K562 cells transfected into pcDNA3.1 plasmid(His-CPEB4 null),the proportion of G0/G1 phase in cell cycle of K562 cells transfected into pcDNA3.1(+)-His-CPEB4 plasmid(His-CPEB4)was increased(P<0.01),the proportion of S phase decreased,cell cycle progression was arrested to G0/G1 phase(P<0.01),the expression level of CDK4(P<0.01),Cyclin D1(P<0.01)protein was decreased,the expression level of P21(P<0.05)was increased;compared with K562 cells transfected into pPLK+Puro plasmid(shRNA-CPEB4 null),the proportion of G0/G1 phase in cell cycle of K562 cells transfected into pPLK+Puro-CPEB4 shRNA plasmid(shRNA-CPEB4)was decreased(P<0.01),the proportion of G2/M phase increased,the cell cycle progression increased(P<0.01),the expression level of CDK4(P<0.01),Cyclin D1(P<0.01)protein was increased,the expression level of P21(P<0.01)was decreased.6.The results of cell migration and molecular protein involved in regulation Compared with K562 cells transfected into pcDNA3.1 plasmid(His-CPEB4 null),the migration ability of K562 cells transfected into pcDNA3.1(+)-His-CPEB4 plasmid(His-CPEB4)was decreased(P<0.01),the expression level of MMP2(P<0.05),MMP9(P<0.05)protein was decreased;compared with K562 cells transfected into pPLK+Puro plasmid(shRNA-CPEB4 null),the migration ability of K562 cells transfected into pPLK+Puro-CPEB4 shRNA plasmid(shRNA-CPEB4)increased(P<0.01),the expression level of MMP2(P<0.05),MMP9(P<0.05)protein was increased.Conclusion:1.CPEB4 inhibited K562 cell proliferation,K562 cell migration,promoted K562 cell apoptosis,and blocked K562 cell cycle progression to G0/G1 phase.2.CPEB4 affects the proliferation,apoptosis,cycle and migration of K562 cells by regulating the expression of AKT,p-AKT,BCL-2,MMP2,MMP9,CDK4,Cyclin D1,P21 and other molecules.