MiR-34a Promotes Apoptosis By Targeting HDAC1 In Acute Myeloid Leukemia Cells

Objective:To investigate the regulation of miR-34 a on HDAC1 and its effect on apoptosis in acute myeloid leukemia cells.Methods:1.We detected the effect of miR-34 a on proliferation and apoptosis of acute myeloid leukemia cells.miR-34 a mimics,miR-34 a inhibitor and miR-34 a scramble were transfused into HL-60 cells.The proliferation and apoptosis of human acute myeloid leukemia cells were observed by regulating the expression of miR-34 a in acute myeloid leukemia cells.2.To investigate the effect of HDAC1 miR-34 a target gene in the process of miR-34 a on proliferation and apoptosis of acute myeloid leukemia cells.Western blot was used to detect the expression of HDAC1 protein after miR-34 a expression changes.3.The double luciferase reporter vector containing HDAC1 3'UTR was constructed,and the action site between miR-34 a and HDAC1 was verified by the double luciferase reporter experiment.Construct the expression vector without HDAC1 3'UTR,and reply to the experiment to verify the interaction between miR-34 a and HDAC1.Results:1.The effect of miR-34 a on proliferation and apoptosis of HL-60 cells.(1)Expression of miR-34 a in HL-60 cells.qRT-PCR detection showed that the expression level of miR-34 a was significantly higher than that of miR-NC Group and Blank group after mimics transfection HL-60 miR-34 a cells(P<0.05).The expression level of miR-34 a inhibitor was significantly lower than that of miR-NC Group and Blank group(P<0.05).There was no statistical difference between Blank group and miR-NC Group(P>0.05)(2)Effect of regulatory miR-34 a on proliferation and apoptosis of HL-60 cell.48 hours after miR-34 a scramble were transfused into HL-60 cells.CCK8 experiment showed that compared with miR-NC Group and Blank group,the cell appreciation rate in miR-34 a group was significantly reduced(P<0.05),and the difference was more significant after 72 and 96 hours.There was no significant difference between Blank group and miR-NC group at each time point(P>0.05).Annexin V-FITC/PI double staining flow cell detection showed that the apoptosis rate of miR-34 a cells after mimics transfection was significantly higher than that of Blank group and miR-NC Group(P<0.05).However,after transfection of miR-34 a cells with HL-60 inhibitor,the apoptosis rate was significantly lower than that of Blank group and miR-NC Group(P<0.05).2 ? The effect of miR-34 a on promoting apoptosis of HL-60 cells and its regulation on HDAC1 expression(1)After transfection of miR-34 a mimics into HL-60 cells,the expression of HDAC1 protein is significantly lower than that of the control group.However,after transfection of miR-34 aa cells with HL-60 inhibitor,the expression of HDAC1 protein is significantly higher than that of the control group.Our date indicates that HDAC1 may be the target of miR-34 a.(2)Connect the 3'UTR region of HDAC1 wild type and mutant type with the double fluorescence reporting vector pmirGLO,and transfect the Recombinant vector with the combination of miR-34 a mimics or miR-34 a Scramble into HL-60 cells,the intensity of fluorescence signals in each group was compared.The results showed that after HDAC1-mut and miR-34 a mimics co-transfection cells,the fluorescence signal value of the cells was statistically significant compared with the control group(miR-NC and HDAC1-mut co-transfection groups)(P>0.05).However,the fluorescence signal value of miR-34 a mimics and HDAC1-wt co-transfection cells was significantly lower than that of the control group(P<0.05).The result revealed that miR-34 a can bind to the 3'UTR region of HDAC1 and inhibit the expression of HDAC1 protein in HL-60 cells.(3)Eukaryotic expression vector pcdna 3.1-HDAC1 without HDAC1 mRNA3'UTR region,and miR-34 a mimics were transfused into HL-60 cells.The resultsshowed that the apoptosis rate of cells decreased significantly(P<0.05).Hower,after miR-34 a mimics were transfected into HL-60 cells,the apoptosis rate increased significantly(P<0.05).This indicated that the transfection of pcdna 3.1-HDAC1 without 3'UTR into HL-60 cells can restore the apoptosis-promoting effect caused by miR-34 a.The miR-34 a play an anti-apoptosis inhibitory role by acting on the binding of HDAC1 3'UTR seed regions.Conclusion:1?The over-expressed miR-34 a in HL-60 of AML cell family showed that the proliferation of cells decreased and the apoptosis rate increased.However,Inhibiting miR-34 a inhibites apoptosis.miR-34 a can promote AML cell apoptosis and play the role of tumor suppressor gene.2?HDAC1 is the target gene of miR-34 a.miR-34 a promotes the apoptosis of HL-60 cells by binding to 3'UTR region of HDAC1 mRNA.