Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, China.
BMC Med Genet. 2013 Jul 19;14:74. doi: 10.1186/1471-2350-14-74.
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). However, expression of miR-663 and its promoter methylation remain status unclear in childhood leukemia.
Promoter 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.
MiR-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.
Expression 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.
越来越多的证据表明，微小 RNA（miRNA）作为异常 DNA 高甲基化机制的靶点具有重要作用。肿瘤抑制 miRNA（如 miR-663）的表观遗传沉默，最近有报道称在几种癌症中因高甲基化而失活，可能在小儿急性髓细胞白血病（AML）中发挥重要作用。然而，miR-663 的表达及其启动子甲基化在儿童白血病中的状态尚不清楚。
通过甲基化特异性 PCR（MSP）和亚硫酸氢盐基因组测序（BGS）检测 miR-663 的启动子甲基化状态。采用半定量和实时 PCR 评估 miR-663 的转录表达，并使用 5-氮杂-2'-脱氧胞苷（5-Aza）去甲基化试剂证实 miR-663 表达与启动子甲基化的关系。
miR-663 在 45.5%（11/24）白血病细胞系中异常甲基化；BGS 显示，在三个 AML 细胞系中启动子明显甲基化；与正常骨髓（NBM）对照样本[10.0%（3/30）]相比，中国小儿 AML 患者[41.4%（29/70）]中 miR-663 的甲基化明显更高。这些结果通过 BGS 和 5-Aza 去甲基化分析得到证实。此外，与对照相比，miR-663 甲基化的 AML 患者和无 miR-663 甲基化的 AML 患者的 miR-663 转录表达均显著降低；然而，miR-663 甲基化的患者与无 miR-663 甲基化的患者在临床特征或法国-美国-英国（FAB）分类方面没有显著差异。
与 NBM 对照相比，miR-663 在小儿 AML 细胞中的表达显著降低；此外，在 AML 细胞系和小儿 AML 样本中均观察到 miR-663 启动子高甲基化的高频率。miR-663 启动子的高甲基化可能受 5-Aza 去甲基化的影响。这些发现表明，miR-663 启动子的高甲基化可能是小儿 AML 发展的早期事件。
