Low Expression of miR-196b Enhances the Expression of <i>BCR-ABL1</i> and <i>HOXA9</i> Oncogenes in Chronic Myeloid Leukemogenesis

<div><p>MicroRNAs (miRNAs) can function as tumor suppressors or oncogene promoters during tumor development. In this study, low levels of expression of miR-196b were detected in patients with chronic myeloid leukemia. Bisulfite genomic sequencing PCR and methylation-specific PCR were used to examine the methylation status of the CpG islands in the miR-196b promoter in K562 cells, patients with leukemia and healthy individuals. The CpG islands showed more methylation in patients with chronic myeloid leukemia compared with healthy individuals (P<0.05), which indicated that low expression of miR-196b may be associated with an increase in the methylation of CpG islands. The dual-luciferase reporter assay system demonstrated that BCR-ABL1 and HOXA9 are the target genes of miR-196b, which was consistent with predictions from bioinformatics software analyses. Further examination of cell function indicated that miR-196b acts to reduce BCR-ABL1 and HOXA9 protein levels, decrease cell proliferation rate and retard the cell cycle. A low level of expression of miR-196b can cause up-regulation of BCR-ABL1 and HOXA9 expression, which leads to the development of chronic myeloid leukemia. MiR-196b may represent an effective target for chronic myeloid leukemia therapy.</p></div

Silencing of <i>CHD5</i> Gene by Promoter Methylation in Leukemia

<div><p>Chromodomain helicase DNA binding protein 5 (CHD5) was previously proposed to function as a potent tumor suppressor by acting as a master regulator of a tumor-suppressive network. <i>CHD5</i> is down-regulated in several cancers, including leukemia and is responsible for tumor generation and progression. However, the mechanism of <i>CHD5</i> down-regulation in leukemia is largely unknown. In this study, quantitative reverse-transcriptase polymerase chain reaction and western blotting analyses revealed that <i>CHD5</i> was down-regulated in human leukemia cell lines and samples. Luciferase reporter assays showed that most of the baseline regulatory activity was localized from 500 to 200 bp upstream of the transcription start site. Bisulfite DNA sequencing of the identified regulatory element revealed that the <i>CHD5</i> promoter was hypermethylated in human leukemia cells and samples. Thus, <i>CHD5</i> expression was inversely correlated with promoter DNA methylation in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates <i>CHD5</i> expression in human leukemia cell lines. <i>In vitro</i> luciferase reporter assays demonstrated that methylation of the CHD5 promoter repressed its promoter activity. Furthermore, a chromatin immunoprecipitation assay combined with qualitative PCR identified activating protein 2 (AP2) as a potential transcription factor involved in <i>CHD5</i> expression and indicated that treatment with DAC increases the recruitment of AP2 to the <i>CHD5</i> promoter. <i>In vitro</i> transcription-factor activity studies showed that AP2 over-expression was able to activate <i>CHD5</i> promoter activity. Our findings indicate that repression of <i>CHD5</i> gene expression in human leukemia is mediated in part by DNA methylation of its promoter.</p></div

<i>CHD5</i> expression is down-regulated in leukemia cell lines and samples.

<p><i>CHD5</i> expression was determined for K-562, KG-1a, HL-60 and Jurkat cell lines by qRT-PCR (top) and western blotting (bottom) (A). Representative results of <i>CHD5</i> mRNA (top) and protein (bottom) expression from ALL, AML and CML patients are presented (B). The distribution of <i>CHD5</i> mRNA (C) and protein level (D) in ALL, AML and CML samples and NMCs (included for comparison) were calculated. For all experiments, β-actin was detected as an internal control. All data are presented as mean ± SD. P<0.001 was considered statistically significant (**).</p

The methylation status of the –560 to –240 region of the <i>CHD5</i> promoter.

<p>The CpG island, located at –560 to –240, was analyzed by BGS. Methylation data from BGS were analyzed by BiQ Analyzer software to generate the lollipop diagram and to calculate the efficiency of bisulfite conversion. Analysis of non-CpG cytosines indicated the efficiency of bisulfite conversion at ∼99%. The lollipop diagram presented the percentage of methylation of each CpG. The overall methylation percentage indicates the total proportion of methylated CpGs in this region taking into account all sequenced alleles.</p

Analysis of CpG island methylation status by MSP.

<p>Six pairwise comparisons were performed, and the significance level was adjusted to 0.0083, in accordance with the Bonferroni correction. CML vs. healthy controls, Fisher's exact test, P = 0.001; AML vs. healthy controls, Fisher's exact test, P = 0.033; ALL vs. healthy controls, Fisher's exact test, P = 0.040; CML vs. AML, Fisher's exact test, P = 0.236; CML vs. ALL, Fisher's exact test, P = 0.135; AML vs. ALL, P = 0.837.</p

Repression of <i>CHD5</i> promoter activity by DNA methylation.

<p>A set of reporter constructs of methylated or mock-methylated fragments of the –560 to –240 region were measured for luciferase activity in K-562 cells. The relative Luc activities of methylated and mock <i>CHD5</i> promoters were normalized to Renilla luciferase activity. All data are presented as mean ± SD. P<0.001 was considered statistically significant (**).</p

The methylation status of <i>CHD5</i> promoter and corresponding <i>CHD5</i> expression in leukemia cell lines treated with DAC.

<p>K-562, Jurkat, HL-60 and KG-1a cells were treated with DNA methyltransferase inhibitor DAC at the indicated concentrations. <i>CHD5</i> expression (A) was analyzed by qPCR (top) and western blotting (bottom). Data are shown as mean ± SD. The methylation status (B) of the –560 to –240 region of the <i>CHD5</i> promoter was analyzed by BGS.</p

Mapping regulatory elements within the <i>CHD5</i> promoter.

<p>The reporter constructs containing the full length (–2000 to –1) <i>CHD5</i> promoter or partial promoter sequences progressively truncated by 200 bp (A) or 100 bp (B) at the 5' or 3' end were measured for luciferase activity in K-562 cells. The promoterless pGL4.10-Basic vector was used as a negative control. The pGL4.10-CMV vector, which uses the CMV promoter, was used as a positive reporter construct. The relative Luc activity of full length and truncated <i>CHD5</i> promoters were normalized to Renilla luciferase activity and compared to normalized Luc luciferase activity from pGL4.10-Basic. All data are presented as mean ± SD. P<0.001 was considered statistically significant (**).</p

Cell functions after transfection and epigenetic drugs.

<p>(A) Protein levels after reintroduction of miR-196b into K562 cells. (B) Proliferation rates of K562 cells after reintroduction or reduction of miR-196b. (C) Cell cycle analysis after reintroduction or reduction of miR-196b. (D) Protein levels after down-regulation of <i>BCR-ABL1</i> and <i>HOXA9</i> by specific siRNAs. (E) Proliferation rates after down-regulation of <i>BCR-ABL1</i> and <i>HOXA9</i> by specific siRNAs. (F) Cell cycle analysis after down-regulation of <i>BCR-ABL1</i> and <i>HOXA9</i> by specific siRNAs. (G) MiR-196b expression in K562 cells after treatment with Aza and PBA separately, or Aza + PBA. (H) BCR-ABL1 and HOXA9 protein levels after treatment with Aza, PBA or Aza + PBA.</p

Primers for construction of reporter constructs.

<p>Primers for construction of reporter constructs.</p