The Clinical Study On Expression And The Methylation Pattern Of MiR-125b In Acute Myeloid Leukemia And Functional Study Of MiR-125b In Myeloid Leukemia Cells

Aims: MicroRNAs play important roles in the initiation and progression of acute myeloid leukemia(AML).This study was aimed to explore the expression status of miR-125 b and the potential clinical relevance in patients with AML,and to study the function of miR-125 b in leukemia cell line.Methods: MiR-125 b transcript level was examined by real-time quantitative PCR(RQ-PCR)in the bone marrow mononuclear cells(BMMNCs)from 33 healthy donors and 114 AML patients.Methylation-specific PCR(MSP)was performed to detect the level of miR-125 b promoter methyaltion in AML patients.Bisulfite sequencing PCR(BSP)was used to verify the density of miR-125 b methylation in representative patients.K562 cells were transfected with miR-125 b overexpressing plasmid or the control vector by liposome transfection.Cell counting and flow cytometry(FCM)were used to measure cell proliferation,survival and apoptosis.The reactivity of K562 cells to decitabine was examined by detecting the inhibitory concentration 50%(IC50)and cell survival assay with decitabine treatment.Results: MiR-125 b was highly expressed in AML patients compared with the health individuals(P<0.01),especially in FAB-M3 subtype.There were no significant differences in sex,hemoglobin(HB),platelets(PLT)and ten common gene mutations between patients with and without miR-125 b overexpression(P>0.05).However,the ages of patients with high miR-125 b expression were younger than the patients with low miR-125 b expression(P=0.05).miR-125 b high-expressed group was associated with both lower white blood cells(WBC)and bone marrow(BM)blast count as compared with miR-125 b low-expressed group(P=0.041 and 0.016,respectively).In addition,significant differences in miR-125 b expression were observed among both FAB/WHO classifications and karyotypes/karyotype classifications(P<0.05).Among AML subtypes of M0-M6,the frequency of miR-125 b overexpression was exceptionally higher in M3 subtype than other subtypes [94%(16/17)versus 29%(28/97),P<0.01].Among the non-M3-AML patients,no significant differences were found between miR-125 b low-expressed and high-expressed patients in sex,age,blood parameters,FAB/WHO classifications,karyotypes/karyotype classifications,and ten gene mutations(P>0.05).Notably,patients with high miR-125 b expression had a higher complete remission(CR)rate than those with low miR-125 b expression(59% versus 34%,P=0.017).However,no definite difference in CR rate between two groups in the cohort of non-M3 patients(P>0.05).Subsequently,Kaplan-Meier analysis showed that AML patients with high miR-125 b expression had significantly longer overall survival(OS)time(median 13 months)than those with low miR-125 b expression(median 6 months)(P=0.046).However,cox regression multivariate analysis showed that miR-125 b expression was not an independent prognostic biomarker in whole-cohort AML patients(P=0.213).Moreover,no significant differences between miR-125 b high-expressed and low-expressed groups in OS were observed not only in non-M3 but also in cytogenetically normal AML(CN-AML)patients(P>0.05).There is no difference in CpG islands methylation between health donors and AML patients.MiR-125 b was unmethylated in both controls and AML patients by MSP,which was further confrmed by BSP in two controls and four AMLpatients(P>0.05).Gene transfection technique was used to obtain stable overexpression of miR-125 b in K562 cellline.The overexpression of miR-125 b could significantly promote the proliferation and survival of K562 cells,while inhibit K562 cells apoptosis in vitro.IC50 and the survival curves of cells after decitabine treatment indicated that miR-125 b does not affect the reactivity of K562 cells to decitabine.Conclusions:(1)MiR-125 b overexpression is a frequent event in AML especially the FAB-M3 subtype,but is not a prognostic indicator in AML.(2)MiR-125 b expression is not regulated by its methylation in AML.(3)The overexpression of miR-125 b promotes the proliferation and survival of K562 cells and inhibits the apoptosis of K562 cells.(4)MiR-125 b does not affect the reactivity of K562 cells to decitabine.