microRNA let-7b在急性淋巴细胞白血病的表达分析与表观遗传学研究

Expression Analysis and Epigenetics of MicroRNA let-7b in Acute Lymphoblastic Leukemia

目的:探讨microRNA let-7b在成人急性淋巴细胞白血病(ALL)的表达及其作用机制,为寻求新的靶向治疗方法提供依据。方法:用甲基化特异性聚合酶链反应(MSP)对ALL患者和非恶性血液病患者骨髓单个核细胞(对照组)中microRNA let-7b启动子区CpG岛的甲基化状态进行分析;应用实时荧光定量聚合酶链反应(q PCR)检测其microRNA let-7b的表达水平;5-氮杂-2'-脱氧胞嘧啶核苷(5-aza-2'-deoxycytidine,5-Aza-d C,DAC)对成人ALL细胞株MOLT-4进行处理;用MSP对药物处理后细胞内microRNA let-7b启动子区CpG岛的甲基化状态进行分析;应用q PCR检测药物处理后细胞内microRNA let-7b的表达水平,探讨microRNA let-7b表达的调控机制。结果:ALL患者microRNA let-7b启动子区CpG岛的甲基化率明显高于非恶性血液病患者,其microRNA let-7b的相对表达量明显降低;5-aza-d C能够显著地抑制MOLT-4细胞株的生长,使细胞周期停滞于G1期,抑制细胞RNA和蛋白质的生物合成,促进细胞发生凋亡,与此同时,能上调microRNA let-7b的表达。结论:在ALL患者中microRNA let-7b的表达受基因组启动子区CpG岛甲基化修饰的调控,microRNA let-7b可能作为抑癌基因,其低表达可能参与ALL的发生、发展,提示microRNA let-7b可能成为治疗ALL的新靶向。

Objective:To study the expression and its mechamisms of microRNA let-7b in adult acute lymphoblastic leukemia(ALL),so as to provide the basis for searching a new targeted therapy.Methods:Firstly,methylationspecific polymerase chain reaction(MSP) was used to analyze the methylation status of CpG islands in microRNA let-7b promoter of bone marrow mononuclear cells in the patients with ALL and patients with non-hematologic malignancies as control,the real-time fluorescence quantitative polymerase chain reaction(qPCR) was used to detect the expression levels of microRNA let-7b in this 2 groups;and then 5-aza-2'-deoxycytidine(5-Aza-dC,DAC) was used to treat ALL cell line MOLT-4;after drug treatment,MSP was used to analyze the methylation status of the CpG islands in microRNA let-7b promoter;the qPCR was used to detect the expression levels of microRNA let-7b,and further explore the regulatory mechanism of microRNA let-7b expression.Results:Hypermethylation of CpG islands in microRNA let-7b promoter in ALL patients was significantly higher than that in patients with non-hematologic malignancies,and the relative expression level of microRNA let-7b was significantly reduced in ALL patients;5-aza-dC could significantly inhibit the growth of MOLT-4 cells and arrest the cells in G_1 phase,thus biosynthesis of RNA and protein was suppressed,and the apoptosis was promoted,meanwhile,5-Aza-dC could increase the expression of microRNA let-7b.Conclusion:In the patients with ALL,the expression of microRNA let-7b is regulated by methylation of CpG islands in the region of genomic promoter.The microRNA let-7b may act as a tumor suppressor,whose low expression is involved in ALL development,indicating the microRNA let-7b may become a new therapeutic target for ALL.