铜离子转运体（CTR1和CTR2）是细胞摄取顺铂的主要转运体，其表达改变可影响肿瘤细胞内药物蓄积，与顺铂耐药密切相关。SNP和miRNA水平改变均可通过影响蛋白表达而影响药物疗效，是具有广阔前景的肿瘤耐药生物标记分子。我们前期小样本研究提示CTR2的rs11794048和rs1128786， CTR1的rs10981694与卵巢癌顺铂耐药相关。基于此，本项目将系统研究CTR1/2多态对卵巢癌顺铂耐药的影响，应用报告基因和EMSA对关联SNP进行功能验证；芯片检测顺铂敏感和耐药卵巢癌组织中miRNA表达差异，结合CTR 3’UTR序列分析，筛选出可能调控CTR表达的miRNA，再用miRNA表达质粒和siRNA转染卵巢癌细胞，MTT测定细胞药物反应性的改变。本项目将SNP和miRNA介导的表观遗传学修饰相结合，系统研究CTR1/2导致卵巢癌顺铂耐药的机制，为卵巢癌耐药筛查和逆转耐药提供新靶点。

Copper transporters (CTR1 and CTR2) are responsible for the uptake of platinum from the extracellular space. Changes in the expression of CTR1 and CTR2 can affect intracellular drug accumulation in tumor, and is closely related with platinum resistance in ovarian cancer. Single nucleotide polymorphism (SNP) and miRNA have effects on drug response by altering protein expression, making them promising biological markers of drug resistance in tumor. Our previous study found that CTR1 polymorphisms were associated with lung cancer platinum resistance, and our small sample studies also suggested that polymorphisms of CTR1 and CTR2 were associated with platinum resistance in ovarian cancer. On this basis, this project will first study the association between the polymorphism of CTR1/CTR2 and cisplatin resistance in ovarian cancer. Then reporter gene system and EMSA experiments will be carried out to verify the function of SNP associated with cisplatin resistance. Thirdly, miRNA expression microarray analysis will be performed to identify characterisedmiRNA with variable expression in drug sensitive or drug resistant patients, then these miRNA will be searched in the database combined with CTR1/CTR2 3 'UTR sequence analysis to screene the miRNA which can bind with the CTR1/CTR2 3 'UTR area. To verify the function of these miRNA, miRNA overexpression plasmid and miRNA interference will be transfected into ovarian cancer cells, and MTT assay will be performed to test changes of cisplatin drug response in the cell. In this study, we aim to systematically study the effect of CTR1/CTR2 in ovarian cancer platinum resistance from genetic variation pathway and miRNA mediated epigenetic modification pathway, and provide new ideas and new targets for ovarian cancer screening and reversion of platinum resistance.

铂类是卵巢癌的一线化疗药物，虽然一线化疗反应率达60-80%，但有55%-75%的患者3 年内复发，5 年生存率仅约30%。化疗耐药是导致治疗失败和复发的最主要原因。因此，查明卵巢癌对铂类化疗药物的耐药机制、建立铂类药物耐药的预警系统对逆转临床耐药、改善患者预后、提高患者5 年生存率具有重要意义。但铂类耐药的机制异常复杂，已有研究在药物在肿瘤细胞内蓄积、肿瘤细胞对药物的转化和解毒功能方面、肿瘤细胞内DNA修复机制、细胞凋亡信号通路等方面都做了很多有意义的探索和研究，但还未取得突变性的进展。铂类进出细胞是铂类发挥疗效的闸门，本项目希望针对这条通路做一些有意义的探索。项目首先对离子转运系统基因多态性及LncRNA H19/IGF2多态性与卵巢癌铂类耐药进行了相关性分析筛选有意义的靶点，发现CTR1 rs10981694 T>G和卡铂的耐药相关，携带G等位基因的患者耐药的风险更大（OR = 4.00, 95% CI 1.31–12.23, p < 0.01）； ATP7A rs2227291 G>C和顺铂的敏感性相关，携带C等位基因的患者对顺铂更敏感（OR=0.4, 95% CI 0.17–0.94, p < 0.03）。H19/IGF2 rs4244809 GG型基因患者发生耐药的风险更低。针对ATP7A rs2227291错义突变构建了野生型和突变型质粒，转染卵巢癌细胞A2780和SKOV3后检测了两种基因型对顺铂IC50的影响，发现转染组显著高于空载对照组，而突变型组敏感性又高于野生型组。CRISPR/Cas9是近几年发展的基因编辑技术，利用本项目的资助我们启动了CRISPR/Cas9全基因敲除文库高通量筛选卵巢癌顺铂敏感基因，前期实验条件的文库病毒包装、病毒感染效率和顺铂作用浓度等方面已经基本稳定，待第一轮的筛选完毕收集细胞提取DNA进行深度测序就可初步评价方法的实用性和研究结果的意义，希望为未来攻克铂类耐药的做出重要贡献。