microRNA在白血病DNMT抑制剂耐药中的作用及机制研究

Resistance to DNA methyltransferase (DNMT) inhibitor develops during the treatment of acute myeloid leukemia (AML) and mechanisms of resistance remain unknown. It is important to identify the mechanisms for improving treatment effect. In our previous study, we established a K562 leukemic line resistant to DNMT inhibitor 5-Aza-2′-deoxycytidine(DAC) and found that oncogenic microRNAs (let-7a-3 and miR-378) were up-regulated due to promoter demethylation. MiR-378 could promote tumor formation and induce the resistance to cytarabine. We will screen the differentially expressed microRNAs and related genes in DAC-resistant K562 using microRNA and cDNA arrays, from which we want to identify the key microRNAs involved in DAC-resistance by regulation of their expression in leukemic cells. The targeting genes of these microRNAs will be identified and the mechanisms involved in the DAC resistance will be further explored. The expression of these microRNAs and the methylation status of their promoters in primary AML cells and the association of them with clinical efficacy and prognosis will be investigated. All this is to explore the feasibility of application of microRNA-targeting interference in reversing DAC resistance and offer new targets for the biotherapy of AML.

DNA甲基转移酶（DNMT）抑制剂在治疗急性髓系白血病（AML）过程中会发生耐药，明确该类药物的耐药机制对于提高其疗效具有重要意义。我们前期研究建立DNMT抑制剂5-氮杂-2′-脱氧胞苷（DAC）耐药的K562白血病株，发现癌性miRNA（let-7a-3和miR-378）发生去甲基化而表达上调，且miR-378能够促进肿瘤形成、增强对阿糖胞苷等的耐药性。本研究拟应用miRNA表达谱和基因表达谱筛选出在DAC耐药株差异表达的miRNA分子和相关基因，通过表达调控进一步明确在DNMT耐药中发挥作用的miRNA；结合靶基因预测及表达调控筛选鉴定该miRNA分子的靶基因，探讨miRNA参与DNMT抑制剂耐药的机制；分析所选miRNA分子在成人AML原代细胞中的表达、甲基化及其临床意义；从而探索应用miRNA分子靶向干预逆转DAC耐药的可行性，为AML的生物治疗提供新靶点。

DNA甲基转移酶（DNMT）抑制剂在治疗急性髓系白血病（AML）过程中会发生耐药，明确该类药物的耐药机制对于提高其疗效具有重要意义。我们建立DNMT抑制剂5-氮杂-2´-脱氧胞苷（DAC）耐药的K562白血病株（K562/DAC），发现K562/DAC细胞中癌性microRNA（miR-378、miR-125等）及癌基因（DDX43、ID1和ID2等）表达上调，而抑癌性miR-186、miR-29b/c等表达下调；.AML临床标本发现AML患者中miR-186、miR-215、miR-218、miR-34c、miR-29b/c表达下调，其低表达患者的总生存期均显著缩短，预示着AML患者不良预后。H19、miR-378和miR-125b在AML中表达上调；H19高表达患者的生存期降低；miR-378高表达患者的无复发生存时间缩短等。DDX43、ID1和ID2表达上调，高表达患者的生存期降低；同时，我们还发现AML患者中H19与ID2表达呈正相关。.体外实验揭示miR-186过表达可使白血病细胞生存降低、凋亡增加；DDX43过表达促进白血病细胞肿瘤球和克隆形成、促进增殖和生存、抑制凋亡、增加对DAC的耐药性，小鼠体内实验揭示DDX43促进白血病形成；通过荧光素酶实验证实miR-186可直接调控DDX43表达。进一步发现K562/DAC中非编码RNA H19启动子发生去甲基化改变而过表达，而DDX43过表达可使K562细胞中H19发生启动子去甲基化、表达水平明显上调；H19干扰可抑制DAC耐药株细胞增殖、生存能力下降。结果提示：miR-186表达下调、靶基因DDX43表达上调、进而诱导H19启动子去甲基化改变而表达增加，导致ID2表达上调，参与DAC耐药。.此外，我们还发现miR-378过表达可促进白血病细胞增殖、集落形成和干细胞克隆球形成增加、抑制细胞凋亡等，其机制可能与下调靶蛋白FUS增加转染细胞干细胞特性，从而促白血病细胞生长有关。在DAC作用下K562细胞miR-29b/c启动子发生去甲基化改变、表达明显上调，ID1启动子的未甲基化态势并未发生改变、ID1表达水平显著下调；而K562/DAC中miR-29b/c启动子区发生去甲基化改变，但其表达水平并未上调，ID1表达却明显上调；提示miR-29b/c诱导的ID1非甲基化依赖的表达异常可能参与DAC耐药。