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

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.

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。

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。本课题组在前期工作中发现还原性叶酸载体（RFC1）编码基因SLC19A1 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。本项目采用荧光素酶报告基因技术，验证了SLC19A1是miR-595的靶基因，rs1051296 位点T＞G多态性显著影响miR-595与SLC19A1的结合；采用蛋白质免疫印迹技术，验证了miR-595显著抑制RFC1蛋白表达；采用ELISA方法，验证了miR-595显著影响进入白血病细胞内的MTX浓度；采用CCK-8法，验证了miR-595显著影响MTX抑制白血病细胞增殖效应；采用流式细胞仪法，验证了miR-595显著影响MTX促白血病细胞凋亡效应和S期细胞比例；采用基质辅助激光解吸电离飞行时间质谱技术，分析了目标SNP基因型，考察了其与MTX化疗疗效、预后和毒性的相关性，发现SLC19A1 rs1051296 TT基因型患儿的复发率（0.00%）显著低于GG/GT基因型患儿（12.50%），无事件生存率（94.74%）显著高于GG/GT基因型患儿（80.21%），GG基因型患儿的血液毒性发生率（13.79%）显著低于GT基因型患儿（38.81%）。总之，本研究阐明了rs1051296位点不同等位基因通过影响miR-595与靶基因的结合能力，调节RFC1表达，导致MTX化疗反应差异形成的分子机制，并在细胞水平和群体水平进行了表型验证。本研究结果从miRNA基因多态性的角度揭示了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了依据。本研究还发现miR-595有望成为MTX化疗敏感性的标志物和逆转MTX耐药的干预靶点，对此进行深入研究，有助于进一步提高MTX化疗的有效性和安全性。