Abstract
BackgroundThere is growing evidence supporting a role for microRNAs (miRNA) as targets in aberrant mechanisms of DNA hypermethylation. Epigenetic silencing of tumor suppressor miRNAs, including miR-663, which has recently been reported to be inactivated by hypermethylation in several cancers, may play important roles in pediatric acute myeloid leukemia (AML). However, expression of miR-663 and its promoter methylation remain status unclear in childhood leukemia.MethodsPromoter methylation status of miR-663 was investigated by methylation specific PCR (MSP) and bisulfate genomic sequencing (BGS). Transcriptional expression of miR-663 was evaluated by semi-quantitative and real-time PCR, and the relationship between expression of miR-663 and promoter methylation was confirmed using 5-aza-2’-deoxycytidine (5-Aza) demethylation reagent.ResultsMiR-663 was aberrantly methylated in 45.5% (5/11) leukemia cell lines; BGS showed that the promoter was significantly methylated in three AML cell lines; methylation of miR-663 was significantly higher in Chinese pediatric AML patients [41.4% (29/70)] compared to normal bone marrow (NBM) control samples [10.0% (3/30)]. These results were confirmed by both BGS and 5-Aza demethylation analysis. In addition, miR-663 transcript expression was significantly lower in AML patients, both with and without miR-663 methylation, compared to controls; however, there were no significant differences in clinical features or French-American-British (FAB) classification between patients with and without miR-663 methylation.ConclusionsExpression of miR-663 was significantly lower in pediatric AML cells compared to NBM controls; furthermore, a high frequency of miR-663 promoter hypermethylation was observed in both AML cell lines and pediatric AML samples. Inactivation of miR-663 by promoter hypermethylation could be affected by 5-Aza demethylation. These findings suggest that hypermethylation of the miR-663 promoter may be an early event in the development of pediatric AML.
Highlights
There is growing evidence supporting a role for microRNAs as targets in aberrant mechanisms of DNA hypermethylation
Our analyses of promoter methylation of miRNAs in pediatric acute myeloid leukemia (AML), using NimbleGen Human DNA Methylation 385 K Promoter Plus CpG Island Arrays, implied that the miR-663 promoter may be hypermethylated in AML (Additional file 1)
The miR-663 promoter is methylated in pediatric AML patients We examined the methylation status of the miR663 promoter in pediatric AML samples and normal bone marrow (NBM)/Idiopathic thrombocytopenic purpura (ITP) control samples
Summary
There is growing evidence supporting a role for microRNAs (miRNA) as targets in aberrant mechanisms of DNA hypermethylation. Epigenetic silencing of tumor suppressor miRNAs, including miR-663, which has recently been reported to be inactivated by hypermethylation in several cancers, may play important roles in pediatric acute myeloid leukemia (AML). Epigenetic tumor suppressor genes is high in AML This suggests that this mechanism has a major role in the development of this rare cancer. The following distinct roles in genomic methylation have been reported for DNMT isoforms: DNMT1 preferentially replicates already existing methylation patterns; DNMT3A and 3B are responsible for establishing de novo methylation Abnormal expression of these methylation-related enzymes may disturb DNA methylation in pediatric AML. A common approach to study DNA methylation is to treat cells with 5-aza-2′-deoxycytidine (5-Aza) demethylation reagent This epigenetic modifier inhibits DNA methyltransferase activity, resulting in DNA demethylation (hypomethylation), as such, treatment with 5-Aza can identify the genes that are activated by methylation
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