ate x2 tests, using SPSS 13.0 software. The results of the luciferase assay and cell proliferation detection assay were analyzed by one-way ANOVA, using SPSS 13.0 software. RT-qPCR analysis Detection of miR-196b followed a similar method mentioned above, except that U6 was used as an internal reference. The primers are shown in Results Methylation of CpG islands in the miR-196b promoter The average expression level of miR-196b was significantly lower in the bone marrow samples from the 16 CML MedChemExpress AZ-6102 patients compared with the 10 healthy controls, as indicated by the RT-qPCR. In light of this, we investigated the role of epigenetic mechanisms that may be involved in the silencing of miR-196b. A CpG island, similar to that present in many tumor suppressor genes, was found in the 1000 bases upstream from the transcription start 12490620 site of miR-196b. The miR-196b CpG island methylation status, in K562 cells treated with 15.55 nM Aza, 1.5 nM PBA, and 15.55 nM Aza+1.5 nM PBA, was examined using BSP. The results indicated that methylation was decreased after all the treatments, particularly after the combined Aza + PBA treatment. Western blot analysis Total protein was prepared and quantified as described in a previous study and then transferred to a polyvinylidene difluoride membrane. Unequal variances were also observed between treatment factors in the miR-196b mimics plus HOXA9 39-UTR group. Again, the results of multiple comparisons showed that miR-196b mimics plus HOXA9 39-UTR caused a decrease in the fluorescence value, which supports the hypothesis that HOXA9 is a target gene of miR-196b. Modulation of BCR-ABL1 and HOXA9 expression by miR196b small interfering RNA and epigenetic drugs Human pre-miR-196b was amplified from normal human bone marrow. Follow up testing was performed on K562 cells infected by 196b virus and pLV virus. A restoration of the expression of miR-196b reduced BCR-ABL1 and HOXA9 protein levels. This was accompanied by a dramatic decrease in the cell proliferation rate and retardation of the G2 stage. A reduction in the expression of miRNA-196b, in the cells where it had been overexpressed, restored BCR-ABL1 and HOXA9 protein levels, enhanced 
cell proliferation , and retarded the S stage of the cell cycle. In addition, down-regulation of BCR-ABL1, by specific siRNAs, reduced BCR-ABL1 protein levels and inhibited proliferation , as also observed in cells that showed over-expression of miRNA-196b and a retarded G1 stage. Down-regulation of HOXA9, by specific siRNAs, reduced HOXA9 protein levels and induced proliferation arrest , as observed in 19497313 cells that showed over-expression of miRNA-196b. This also resulted in the retardation of the G1 stage of the cell cycle. Promoter methylation can be reversed by specific epigenetic drugs. We treated K562 cells with Aza, PBA and Aza + PBA, which resulted in partial but efficient demethylation of the miR196b promoter. The miR-196b expression was restored in K562 cells that were treated with Aza + PBA but not in those treated with Aza and PBA, separately. The three treatment groups showed a significant reduction in both BCR-ABL1 and HOXA9 protein levels. vs. miR-196b mimics control+BCR-ABL1-39-UTR, P = 0.006; vs. miR-196b mimics+ BCR-ABL1-39-UTR -mutant, P,0.001; vs. lipo2000+ BCR-ABL1-39-UTR, P,0.001; vs. lipo2000+BCR-ABL1-39-UTR -mutant, P = 0.001. vs. miR-196b mimics contro+HOXA9-39-UTR, P,0.001; vs. miR-196b mimics+ HOXA9-39-UTR -mutant, P = 0.016; vs. lipo2000+ HOXA9-39-UTR, P = 0.02