miR-SNP介导卡培他滨结直肠癌化疗反应个体差异的分子机制研究

Capecitabine is the first-line treatment for advanced colorectal cancer. However, individual response to capecitabine chemotherapy is obvious, and the molecular mechanisms are largely unknown. In this project, based on our preliminary findings of capecitabine chemotherapy response related miR-SNP, focusing on the molecular mechanism of individual response to capecitabine chemotherapy, taking rs7911488 of miRNA-1307 as the enter-point, and using DNA sequencing method, real-time PCR, western blotting, fluorescent reporter gene analysis, MTT assay, and clinical evaluation on animal models, we aimed to investigate the molecular mechnism that rs7911488 through impacting miR-1307 expression levels, thus affecting the expression of transporters and metabolic enzymes, eventually results in individual response to capecitabine chemotherapy. Furthermore, we will in vitro and in vivo study the effect of intervention of miR-1307 on the response to capecitabine chemotherapy. These findings could faciliate understanding the impact of genetic variation in chemotherapy response to capecitabine, guiding rational use of capecitabine for clinical chemotherapy, as well as laying the foundation for designing treatment way to improve the sensitivity to capecitabine chemotherapy regarding miR-1307 as targets.

卡培他滨是晚期结直肠癌化疗的一线用药；但其疗效个体差异显著，确切机制尚待研究。本项目拟在前期研究发现卡培他滨化疗反应相关miR-SNP的基础上，围绕卡培他滨化疗反应个体差异形成机制这一科学问题，以miRNA-1307 rs7911488为切入点，从临床样本、肿瘤细胞株及动物模型三个层面，采用测序法、Real-time PCR法、Western blotting法、荧光报告基因分析、MTT法、动物模型疗效评价等技术，研究rs7911488通过影响miR-1307表达水平，以调节转运体/代谢酶表达，从而导致卡培他滨化疗反应个体差异形成的具体分子机制及干预miR-1307表达或功能对卡培他滨化疗敏感性的影响。为深入了解miRNA遗传变异对卡培他滨化疗疗效的作用机制、指导临床化疗合理用药，及设计以miR-1307为靶标提高化疗敏感性的治疗途径奠定基础，研究具有显著的理论创新性和潜在的临床应用价值。

目的背景：研究在结肠直肠癌（CRC）中药物转运蛋白基因上microRNA（miRNA）结合靶点内的单核苷酸多态性与5-FU/卡培他滨化疗疗效的相关性。探讨miRNA的结合位点内的单核苷酸多态性与晚期结肠癌卡培他滨化疗疗效的相关性。研究内容：1.ABCC4 3’-UTR上miR-3190-5p结合靶点内的基因多态性影响结直肠癌化疗疗效的机制研究；2.Pre-mircoRNA末端环内基因多态性rs7911488影响结直肠癌化疗疗效的机制研究；3.ICOS/CD28-ICOSL通路基因多态性与晚期结肠癌卡培他滨化疗疗效和不良反应的相关性研究；4.miR-SNP与结肠癌化疗相关性研究。重要结果和关键数据：1.在结直肠癌中ABCC4 3'-UTR上多态性位点rs3742106 T/T基因型患者药物反应率明显高于G/G和G/T基因型；2.在结直肠癌患者中pre-miR-1307上多态性位点rs7911488 T>C与卡培他滨化疗疗效显著相关。T/T、T/C和C/C基因型患者对卡培他滨的化疗反应率的分别为44.35%（55/124）、51.33%（58/113）和24.32%（9/37）；3.（1）rs744591和rs745666与结直肠癌患者卡培他滨联合化疗的疗效显著相关，（2）rs2114358、rs35770269和rs73239138基因型与卡培他滨化疗不良反应的发生显著相关；4.(1) 统计分析发现ICOS rs4404254和ICOS rs1559931与化疗疗效有显著相关性，(2)三个miRSNPs （ICOSL rs15927、ICOSL rs3804033和CD28 rs3181113）与化疗不良反应的发生密切相关。科学意义：1.在结直肠癌中ABCC4 3'-UTR上多态性位点可望成为一个生物标志物，指导结直肠癌中5-FU和卡培他滨化疗的个体化用药。2.pre-miR-1307 rs7911488 C等位基因会导致miR-1307-3p下调和TYMS上调，引起结直肠癌中卡培他滨化疗的不敏感性。3.接受卡培他滨治疗的晚期结肠癌患者中，rs744591和rs745666与化疗疗效显著相关，rs2114358、rs35770269和rs73239138与不良反应发生显著相关。这对预测结肠癌患者卡培他滨化疗疗效具有潜在价值。