Circ_0062018作为ceRNA调节甲氨蝶呤敏感性的机制研究

Methotrexate (MTX) resistance is a great challenge in the treatment of childhood acute lymphoblastic leukemia (ALL). In our previous studies, it was found that miR-595 induced MTX resistance by inhibition of reduced folate carrier 1 (RFC1). There was significant down regulation of circ_0062018 which might have strong interactions with miR-595 in MTX-resistant samples. However, it was unclear whether circ_0062018 could enhance MTX sensitivity via functioning as miR-595 sponges and increasing expression of RFC1. The following work will be carried out in our research. Firstly, we will test whether overexpression and knockdown of circ_0062018 lead to changes of MTX sensitivity in leukemia cells. Secondly, we will explore the underlying molecular mechanisms of circ_0062018 as ceRNA in the regulation of MTX sensitivity, using techniques such as fluorescence in situ hybridization, co-immunoprecipitation, and luciferase reporter gene assays. Thirdly, the feasibility of circ_0062018 as a biomarker for MTX sensitivity and its potential of overcoming MTX resistance will be validated in ALL population and animal model. This study will help to disclose potential reasons for individual differences of MTX sensitivity from the perspective of nonconding RNA-mediated regulation of RFC1 acitivity, and to improve the diagnosis and treatment in refractory/relapsed ALL.

甲氨蝶呤（MTX）耐药是急性淋巴细胞白血病（ALL）临床治疗中常见的难题。我们在前期工作中发现miR-595通过抑制还原性叶酸载体（RFC1）表达，导致MTX耐药，而与miR-595有较强结合作用的circ_0062018在MTX耐药样本中的表达显著下调，circ_0062018是否可通过吸附miR-595，调节RFC1表达，影响MTX敏感性尚不清楚。本项目拟在细胞水平验证circ_0062018表达对MTX敏感性的调节作用；采用荧光原位杂交、免疫共沉淀、荧光素酶报告基因等技术，阐明circ_0062018作为ceRNA调节MTX敏感性的分子机制；在人体组织和实验动物水平验证circ_0062018作为MTX敏感性标记物和逆转耐药干预靶点的可行性。本研究将有助于从非编码RNA介导的RFC1活性调控角度，揭示MTX敏感性个体差异的潜在原因，对于提高难治/复发ALL诊疗水平具有重要的临床意义。

甲氨蝶呤（MTX）耐药是急性淋巴细胞白血病（ALL）临床治疗中常见的难题。我们在前期工作中发现miR-595通过抑制还原性叶酸载体（RFC1）表达，导致MTX耐药，而与miR-595有较强结合作用的circ_0062018在MTX耐药样本中的表达显著下调，circ_0062018是否可通过吸附miR-595，调节RFC1表达，影响MTX敏感性尚不清楚。本项目在BALL-1细胞层面开展了表达动力学实验，考察了系列递增浓度MTX对RFC1 mRNA（SLC19A1）、miR-595和circ_0062018表达的影响；在细胞功能研究方面考察了MTX耐药细胞与对照细胞中SLC19A1、miR-595和circ_0062018的表达差异以及过表达circ_0062018对细胞增殖和MTX敏感性的影响；在机制研究方面考察了过表达circ_0062018对miR-595和RFC1 mRNA和蛋白表达的影响。在ALL人群层面，通过分析骨髓组织中的Circ_0062018表达与MTX化疗敏感性、ALL疗效以及ALL生存预后的相关性，考察了Circ_0062018对MTX敏感性、ALL疗效和预后的预测价值。另外本项目还基于MTX耐药细胞株，构建了SLC19A1 ceRNA网络，在ALL组织水平进行表达相关性分析和临床意义验证，筛选出多个与MTX化疗敏感性、ALL预后显著相关的非编码RNA生物标记物。本项目的研究结果有助于从非编码RNA介导的RFC1活性调控角度，揭示MTX敏感性个体差异的潜在原因，对于提高难治/复发ALL诊疗水平具有重要的临床意义。