| Background and ObjectiveAcute myeloid leukemia(AML)is a heterogeneous disease in biology and in clinical matters.In the world,China ranks third in the incidence of AML and China has the highest levels of mortality in young patients with AML.The pathogenesis of AML is still not fully understood and thus hampers the development of new treatments for AML.MicroRNAs(miRNAs)are a novel short,non-coding RNA molecules that regulate gene expression by targeting the 3'-untranslated region(3'-UTR)of the mRNA and leads to the inhibition of translation or degradation of mRNA.Thus,miRNAs may be regarded as native regulators of gene expression.The latest study has proved that miRNAs are potential and promising tools for cancer diagnosis and treatments.Some studies showed that miR-125 b is associated with leukemia.We transfected miR-125 b mimics into Kg1 a and HL60 cells,to investigate the biological role of miR-125 b in AML,the possible signal pathway which miR-125 b was involved and the potential molecular mechanism involved in this process an,provide new therapeutic target for AML.Methods1.Different dose of synthetical miR-125 b mimics and the corresponding negative control(miR-NC)were transfected into Kg1 a and HL60 cells,make miR-125 b overexpress in Kg1 a and HL60 cells.2.The expressions of mi R-125 b were assessed by qRT-PCR in Kg1 a and HL60 cells treated with miR-125 b mimics.3.The propagation of the Kg1 a and HL60 cells was evaluated by MTT assay after transfered of miR-125 b mimics.4.Cell apoptosis population and cell cycle detection were analysed by Annexin v-FITC/PI flow cytometry.5.The expression of I?B-? phosphorylation and I?B-? was determined using western blotting in miR-125 b transfected Kg1 a and HL60 cells.6.The effect of miR-125 b on p65 distribution in both nuclear extracts and cytoplasmic extracts was detected by western blotting in miR-125 b transfected Kg1 a and HL60 cells.7.The expression of NF-?B-regulated genes involved in cell cycle arrest(cyclinB,cdc-2,mdm-2)and apoptosis(Bcl-2,p53,Bax)was assessed by by western blotting in miR-125 b transfected HL60 cells.8.All experiments were performed with at least three independent replicates.The results were analyzed using Student's t-test for comparisons between two groups and Analysis of Variance(ANOVA)was used to evaluate the differences among three or more groups.A P value < 0.05 was considered to be statistically significant.Results1.To evaluate the efficiency of miR-125 b mimics,an qRT-PCR assay was performed to determine the expression level of miR-125 b in Kg1 a and HL60 cells transfected with miR-125 b mimics or its corresponding negative controls.qRT-PCR results showed that the level of miR-125 b increased significantly following transfection with miR-125 b mimics but not two control groups(P<0.001),but there was no difference between two control groups(P>0.05).2.The role of miR-125 b in cell growth was a determined by MTT assay.MTT assay results showed that miR-125 b mimics transfection group had reduced cell viability in a miR-125 b concentration dependent manner compared with two control groups(P<0.01),but there was no difference between two control groups(P>0.05),indicating that miR-125 b may inhibit the proliferation of Kg1 a and HL60 cells.3.The effects of miR-125 b on cell invasion of Kg1 a and HL60 cells were determined by Matrigel invasion assay as well.The results showed that,migration of Kg1 a and HL60 cells was inhibited in a mi R-125 b concentration dependent manner when compared with two control groups(P<0.05),but there was no difference between two control groups(P>0.05).4.To further explore the mechanisms involved in the inhibition of mi R-125 b on AML cells proliferation,the effects of miR-125 b on cell cycle and cells apoptosis was assessed by flowcytometry using Annexin-FITC/PI kit.Cell cycle detection results showed that there was a concentration dependent accumulation of cells in the G2/M-phase of the cell cycle in the miR-125 b mimics transfection group.And miR-125 b dose-dependently increased cell apoptosis rate 48 h after transfection.There results indicate that overexpression of miR-125 b could arrest the cell cycle at the G2/M phase in AML cells.5.To determine whether miR-125 b inhibited AML cells proliferation and promoted cell phase arrest by targeting the NF-?B signaling pathway,the expression of I?B-? phosphorylation,I?B-?,nuclear p65 and cytoplasmic p65 was determined using western blotting.Western blotting results showed that p-I?B-? and nuclear p65 were significantly increased after TNF-? treatment,while I?B-? degrade and cytoplasmic p65 was decreased simutaneously.However,transfection with miR-125 b mimics before stimulated with TNF-? suppressed the increase of p-I?B-? and nuclear p65 expression as well as I?B-? degradation and the decrease of cytoplasmic p65 induced by TNF-? in a concentration dependent manner in both Kg1 a and HL60 cells compared with control groups(P<0.05).6.NF-?B is known to regulate the expression of numerous proteins involved in cell cycle arrest and apoptosis.Western blotting results showed that miR-125 b inhibited the expression of cyclin B,cdc-2,mdm-2 and in a dose-dependent manner in HL60 cells compared with control groups(P<0.05).In addition,the effect of miR-25 b on the NF-?B-dependent gene products that are involved in cell apoptosis were determined as well.MiR-125 b but not mi R-NC promoted the expression of apoptosis-related protein Bax and p53,while suppressed the expression of Bcl-2(P<0.05).CONCLUSION1.The datas suggested mi R-125 b may suppress human AML cells proliferation and invasion,induce G2/M phase cell cycle arrest and promote cells apoptosis in vitro.2.The present work certificated that miR-125 b can significantly inhibit human AML cells proliferation and promotes cells apoptosis by targeting NF-?B signaling pathway.3.Our findings provided evidence that miR-125 b can be viewed as an promising therapeutic target for AML. |
PDF Full Text Request