microRNA-21在慢性髓细胞白血病耐药机制中作用的研究

The application of imatinib, the first-generation of BCR-ABL tyrosine kinase inhibitor (TKI), for chronic myeloid leukemia (CML) therapy is a milestone of tumor targeted therapy. However, in newly diagnosed patients with chronic phase CML, the rate of resistance to imatinib was up to 20%, increasing to 70%～90% for patients in the accelerated/blastic phase. Recent study suggested a correlation between overexpression of microRNA-21 (miR-21) and drug resistance in CML cells. But few researches have been performed to confirm it. Our previous work constructed three pairs of miR-21 targeting TALENs and also found out an effective way to transduce them into suspension cells. Knockout clones were isolated through eGFP enrichment, PCR screening, and microRNA quantification. Base on these, here we aim to delete miR-21 in CML cell lines K562, K562/Adr, K562/G01 and 32Dp210T315I by using TALEN, respectively. Further analyzing single-cell-derived miR-21 knockout clones, we expect to find the phenotype and genotype difference between the parental wild type and the miR-21 knockout cells, which may disclose the function of miR-21 in CML drug resistance.

第一代TKI伊马替尼治疗慢性髓细胞白血病(CML)是肿瘤靶向治疗的典范。但仍有20%的病人在起始治疗4年内对其耐药，一旦疾病进展，耐药率更高达70～90%。近年来，有学者提出miR-21在CML细胞中高表达可能与其耐药有关，但目前相关研究很少。我们的前期研究构建了三对可在基因组水平上靶向敲除miR-21基因的TALEN质粒，探索出了向悬浮细胞中转染TALEN质粒并有效筛选基因敲除的单细胞克隆的方法。在此基础上，本项目拟通过TALEN介导，分别在敏感或耐药的CML细胞株K562、K562/Adr（KAR）、K562/G01和32Dp210T315I中敲除miR-21基因，筛选、培养miR-21敲除的单细胞克隆。通过比较野生型和miR-21敲除的单细胞克隆在细胞表型及基因型上的差异，研究miR-21和ABL激酶区突变的关系和它在CML耐药机制中的作用，为CML治疗新靶点提供思路。

第一代TKI伊马替尼治疗慢性髓细胞白血病(CML)是肿瘤靶向治疗的典范。但仍有20%的病人在起始治疗4年内对其耐药，一旦疾病进展，耐药率更高达70～90%。近年来，有学者提出miR-21在CML细胞中高表达可能与其耐药有关。运用CRISPR/Cas9介导的基因工程技术，我们分别建立了miR-21敲除的K562、KAR、K562/G01细胞的单细胞克隆（3个克隆/细胞株）。实验证明miR-21基因敲除使得K562和K562/G01细胞增殖变缓、凋亡加速，发现miR-21敲除的K562 单细胞克隆中P-AKT和PI3K显著下调，同时BCR-ABL和p-BCR-ABL的表达也是明显下调的，miR-21敲除的K562细胞对慢性粒细胞白血病靶向治疗药物达沙替尼的敏感性增加，对伊马替尼的敏感性没有明显变化。