还原性叶酸载体miRNA靶序列基因多态性对甲氨蝶呤化疗反应的影响及其分子机制研究

项目名称： 还原性叶酸载体miRNA靶序列基因多态性对甲氨蝶呤化疗反应的影响及其分子机制研究

项目编号： No.81503135

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 医药、卫生

项目作者： 王淑梅

作者单位： 首都医科大学

项目金额： 17.9万元

中文摘要： 甲氨蝶呤（MTX）是儿童急性淋巴细胞白血病（ALL）的重要化疗药物，其治疗窗窄，毒性大，药代和药效的个体间差异明显，具体机制尚不清楚。我们在前期工作中发现还原性叶酸载体基因3’端非翻译区的miR-595结合位点rs1051296多态性与ALL患儿的MTX血药浓度相关。本项目拟采用荧光实时定量PCR、蛋白质免疫印迹、荧光素酶报告基因等技术，研究不同等位基因通过影响miR-595与靶基因的结合能力，调节目标蛋白表达，导致MTX化疗反应差异形成的分子机制；然后体外分离培养骨髓白血病细胞，分析不同基因型对MTX摄取率和白血病细胞敏感性的影响；最后选择不同基因型的ALL患儿，验证基因型与MTX群体药代动力学、化疗疗效和毒性的关系。本研究将有助于从miRNA基因多态性的角度揭示MTX化疗反应个体差异的潜在原因，为临床根据基因型调整MTX个体化给药方案提供依据，进一步提高MTX化疗的有效性和安全性。

中文关键词： 甲氨蝶呤；还原性叶酸转运载体；微小RNA；单核苷酸多态性；群体药代动力学

英文摘要： Methotrexate (MTX) was an important chemotherapeutic drug for childhood acute lymphoblastic leukemia (ALL). It exibited narrow therapeutic window, profound toxicities and notable inter-individual differences in pharmacokinetics and pharmacodynamics, but the exact mechanism remained unclear in children with ALL. In our previous studies, it was found that the miRNA-595 binding site polymorphisms (rs1051296) in 3'-untranslated region of the reduced folate carrier (RFC) gene were significantly associated with the blood concentration of MTX in ALL children. The following work will be carried out in our research. Firstly, the effects of rs1051296 on “miRNA - RFC gene” interaction and the expression of RFC will be explored in the present study, using techniques such as real-time fluorescent quantitative PCR, Western blotting and luciferase reporter gene, to illustrate the underlying mechanisms of the associations between the investigated single nucleotide polymorphism (SNP) and the difference in MTX chemotherapy response. Secondly, myeloid leukemia cells of children with ALL will be isolated and cultured in vitro studies, to analyze the effects of different genotypes on MTX uptake rate and the sensitivity of leukemia cells to MTX. Finally, ALL children carrying different genotypes will be selected to verify the associations between the studied genotype and population pharmacokinetics, efficacy and toxicities of MTX chemotherapy. This study will help to disclose potential reasons for individual difference of MTX chemotherapeutic responses from the perspective of miRNA genetic polymorphisms, provide theoretical basis for clinically individualized dosing regimen of MTX adjusted by genotypes, and the efficacy and safety of MTX chemotherapy can be further improved thereby.

英文关键词： Methotrexate;reduced folate carrier;microRNA;single nucleotide polymorphism;population pharmacokinetics