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Objective: To study the expression level and CpG island methylation status of miR-34b in leukemia cell lines and to research the effect of DNA methylation on the proliferation of leukemia cells regulated by miR-34b., Method: Taqman real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was carried out to detect the relative expression of miR-34b in control group (bone marrow cells of 20 children without blood disease) and 8 leukemia cell lines (U937, HL-60, MV4-11, M2R, K562, Raji, CCRF, DAMI). Methylation-specific polymerase chain reaction (MSP) was carried out to detect the methylation differences of miR-34b in control group (Bone marrow cells of 23 children without blood disease), 8 leukemia cell lines. HL-60 and K562 were treated with methyltransferase inhibitor (5-aza-2-dC) for further detection of its methylation status and expression of miR-34b. Hsa-miR-34b mimics was transfected into K562 cell by liposome, the transfection efficiency was detected by flow cytometry. The cell proliferation of hsa-miR-34b transfected group in each stage was measured with CCK-8 assay, and then compared with non-transfected group and negative control group., Result: The relative expression level of miR-34b in the group of children without blood disease and the group of leukemia cell lines were 5.22 ± 1.15, 0.03 ± 0.03. The results showed that, the group of leukemia cell lines was significantly different from the control group (t = 4.538, P < 0.01) . Eight leukemia cell lines showed methylation, the positive rate of the methyl was 100%. There was no methylation in the 23 cases of control group. After leukemia cell lines HL-60 and K562 were treated with 5-aza-2-dC, the methylated bands became obviously weakened, and the relative expression levels of miR-34b substantially increased 49.5 times and 18.8 times respectively. After hsa-miR-34b mimics was transfected into K562 cell by liposome, its transfection efficiency detected by flow cytometry was 61% and the cell proliferation was measured with CCK-8 assay from which it was found that the cell proliferation was significantly suppressed compared with the control group at 48 h (t = 9.303, P < 0.01), 72 h (t = 65.617, P < 0.01), 96 h (t = 36.878, P < 0.01) and 120 h (t = 18.748, P < 0.01) in hsa-miR-34b transfected group, with the inhibition rate of 12.2% (48 h), 45.7% (72 h), 32.5% (96 h) and 22.9% (120 h)., Conclusion: The hypermethylation of promoter leads to decrease in the expression levels of miR-34b in leukemia cell lines, which attenuate mechanism of proliferative inhibition may be one of the reasons of occurrence or development of childhood leukemia.