DNA甲基化介导的miR-720沉默与急性髓系白血病生物学特征的关系  

DNA methylation-mediated epigenetic silencing of miR-720 contributes to leukemogenesis in acute myeloid leukemia

作　　者：唐善浩[1] 裴仁治[1] 李空飞[1] 马俊霞[1] 张丕胜[1] 陆滢[1] 刘旭辉[1] 杜小红[1] 陈冬[1] 沙科娅[1] 曹俊杰[1] 李双月[1] 

机构地区：[1]宁波大学医学院附属鄞州医院血液科,315040

出　　处：《中华血液学杂志》2014年第11期1009-1012,共4页Chinese Journal of Hematology

摘　　要：目的 探讨miR-720在急性髓系白血病（AML）患者中的表达水平、调控机制及其与白血病肿瘤生物学行为的关系.方法 采用荧光定量PCR检测38例AML患者和20名正常对照骨髓细胞miR-720表达水平;采用焦磷酸测序法定量检测AML患者和正常对照miR-720启动子甲基化水平;构建慢病毒介导的miR-720过表达kasumi-1细胞,采用流式细胞术、WST-1法、集落形成实验、Transwell细胞迁移实验及Western blot法分别检测miR-720对kasumi-1细胞增殖、凋亡、细胞周期、集落生成、迁移及P53介导的细胞凋亡通路的影响.采用重亚硫酸盐测序法检测地西他滨处理前后kasumi-1细胞miR-720甲基化水平,采用荧光定量PCR检测地西他滨处理后kasumi-1细胞miR-720表达水平.结果 与正常对照相比,AML患者miR-720表达水平明显降低（0.69±0.09对3.00±0.46,P＜0.01）,且miR-720启动子区域呈现显著高水平的DNA甲基化[（75.56±2.35）％对（47.65±2.78）％,P＜0.01].miR-720过表达的kasumi-1细胞自身凋亡率明显增高（P=0.017）,对细胞毒药物依托泊苷的凋亡敏感性也明显增加（P=0.004）,细胞增殖明显受抑（P＜0.01）,细胞集落形成能力减弱（P=0.005）,细胞周期阻滞于G1/G0期,细胞迁移能力下降.miR-720的过表达显著诱导kasumi-1细胞P53凋亡相关蛋白的表达包括P53、Bax以及诱导NF-κB信号转导通路的激活.地西他滨处理后的kasumi-1细胞miR-720表达水平较处理前明显增高,而启动子甲基化水平明显降低，结论 AML患者骨髓细胞miR-720表达水平明显降低,DNA甲基化介导的miR-720表达抑制有助于白血病生物学特征的维持。Objective To investigate the expression level and regulation mechanism of miR-720 as well as the association of miR-720 expression with leukemia biological characteristics.Methods Expression and promoter methylation of miR-720 were determined by quantitive PCR and pyrosequencing in 38 patients with AML and 20 normal controls.Lentivirous-mediated miR-702 overexpression was constructed in AML cell line kasumi-1.The cell proliferation,apoptosis,cycle,colony formation,migration and P53-mediated apoptosis pathway were determined.Results AML patients showed significantly lower miR-720 expression compared with normal controls （0.69 ± 0.09 vs 3.00 ± 0.46,P＜0.01）; The methylation level of miR-720 promoter region in AML patients were significantly higher than normal controls [（75.56± 2.35）％ vs （47.65 ± 2.78）％,P＜0.01].miR-720 overexpression in kasumi-1 cells induced significantly increased cell apoptosis （P=0.017）,elevated apoptosis sensitivity to etoposide （P=0.004）,and reduced cell proliferation （P＜0.01）.miR-720 overexpression also induced reduced colony formation （P=0.005）,cell cycle arrest in G1/G0 phase and decreased migration ability in kasumi-1 cells.In addition,overexpression of miR-720 significantly induced increased cell apoptosis-related proteins including P53 and Bax,and activation of NF-κB signal transduction pathway.After kasumi-1 cells were treated with 1 uM decitabine for 48 hours,miR-720 promoter methylation reduced significantly,and miR-720 expression significantly increased.Conclusion The expression of miR-720 in AML patients reduced significantly,and DNA methylation-mediated epigenetic silencing of miR-720 contributed to maintain the malignant characteristics of AML.

关 键 词：白血病 髓样 急性 微RNAS DNA甲基化 肿瘤特征 地西他滨 

分 类 号：R733.7[医药卫生—肿瘤]