Effects Of Antisense MiRNA-125b And MiRNA-27a Mimics On HL-60 Cells For Resistance

OBJECTIVE: The miRNA-27 a and antisense miRNA-125 b mimics were transfected into HL-60 daunorubicin-resistant cells to observe the effects of these two mimetics on the resistance of daunorubicin(DNR)to HL-60 cells.METHODS:1.A small dose of DNR induced acute promyelocytic leukemia cell line HL-60 for one month to produce DNR drug resistance.2.The antisense strand mimic of miRNA-125 b and the sense strand mimic miRNA-27 a and negative control(N.C.)were transfected into HL-60/DNR-resistant cells by liposome Endo Fectin TM-MAX.3.Observe the transfected cell status by fluorescence microscopy after incubation for 48 hours.extract the RNA by Ta Ka Ra RNA extraction kit(RNAiso Plus),verify the transfection according to All-in-One ? miRNA q RT-PCR kit which was provided by Gene Copoeia TM,and the miRNA primer sequences were provided by Shanghai Bioengineering Co.,Ltd..4.With low doses of daunorubicin acting on the above cells,MTT assay was performed to the cells after 48 and 72 hours.Observe whether these cells are resistant to DNR.Results: 1.The state of HL-60 sensitive cells: there were a large area of cell debris in the first 3 days under the microscope,then we did not find significant cell debris after a month,confirmed that HL-60 cells significantly improved tolerance to DNR,cells had been resistant to DNR;2.Evalue the efect by fluorescence microscope: Transfected HL-60/DNR-resistant cells with miRNA mimics and then incubated for 48 hours,there were exsist fluorescence in large number of cells,indicated that the mix of miRNA mimic complex had been expressed intracellularly;3.The results of real-time quantitative PCR showed that the miRNA-27 a and antisense miRNA-125 b were significantly increased,compared with the cells which was transfected with negative control(P <0.05).4.The results of MTT assay showed that the inhibitory rate of daunorubicin on HL-60 / DNR-resistant cells transfected with miRNA-27 a mimics was significantly higher than that of the miRNA-NC transfected group(P<0.05),the inhibitory rate of HL-60 / DNR cells transfected with antisense miRNA-125 b was not significantly changed(P> 0.05).Conclusion: Upregulation of miRNA-27 a can decrease the resistance of HL-60 / DNR-resistant strains to daunorubicin,and it is proved that the overexpression of antisense miRNA-125 b can not reverse the resistance of HL-60 / DNR-resistant strains to daunorubicin.