The Roles Of MicroRNA-128a Targeted Bmi-1 In The Proliferation And Apoptosis Of Myeloid Leukemia Cells

Objective: To clarify the mechanism of miR-128 a targeting Bmi-1 on proliferation and apoptosis of myeloid leukemia cells.We examined the differential expression of miR-128 a and Bmi-1 in myeloid leukemia cell lines,which may provide a new treatment strategy for leukemia.Methods:1.The leukemia cell lines including human chronic myeloid leukemia K562 cell,human acute monoblast leukemia THP-1 cell and human acute myeloid leukemia KG1 cell were chosen as the researching object.The expression of Bmi-1 in those cells and normal human mononuclear cells were detected by real-time PCR.2.Using network prediction softwares to forecast the target gene of Bmi-1 and the differential expression of miR-128 a in leukemia cells(human chronic myeloid leukemia K562 cell line,human acute myeloid leukemia KG1 cell line and human acute monoblast leukemia THP-1 cell line)and normal human mononuclear cells were tested by qRT-PCR.3.The relationship of miR-128 a and Bmi-1 were verified by using bioinformatics technology and dual luciferase experiment.4.Using qRT-PCR to detect the over-expression miR-128 a in three leukemia cells(K562 cells,THP-1 cells and KG1 cells)and the expression of Bmi-1 in those cells were tested by western blot.5.MTT,colony formation,Immuno-fluorescence staining assays were conducted to verify the changes of proliferation and self-renewal ability of three leukemia cells in vitro after upregulation of miR-128 a in K562 cells,THP-1 cells and KG1 cells.6.Flow cytometry and western blot assays were used to determine the capacity of apoptosis of leukemia cells(K562 cells,THP-1 cells and KG1 cells).Results:1.Bmi-1 was highly expression expressed in three leukemia cell line(K562,KG1,THP-1).2.Based on the network prediction software(Targetscan Human 7.1,miRanda and microRNA.org),miR-128 a was the common target of Bmi-1,and miR-128 a was found to be significantly down-regulated in three leukemia cell lines(K562cells,KG1 cells,THP-1cell)versus its parental line of normal mononuclear cells.The expression of Bmi-1 was inversely correlated with miro-128 a in leukemia cells.3.Bmi-1 was a direct target of miR-128 a,which was confirmed by dual-luciferase assays and bioinformatics(Targetscan Human 7.1,miRanda and microRNA.org).4.Construction of the siRNA plasmid sequence miR-128 a mimic which were designed by biotechnology company were transiently transfected into three leukemia cells that over-expression of miR-128 a resulted in reduction of the expression of Bmi-1 and the capability of cell proliferation respectively compared with control cells transfected miR-128 a NC.5.The ability of colony formation and self-renewal were attenuated after transfection with miR-128 a mimic in three leukemia cell lines.6.Flow cytometry analysis and western blot showed that over-expression of miR-128 a promoted the capability of three leukemia cells apoptosis.Conclusion:1.The expression of miR-128 a and Bmi-1 in human leukemia cell lines were significantly different and negatively correlated.2.MiR-128 a might mediate the progression of leukemia through negative regulation of the expression of Bmi-1.