circRNA_0001658/DLK1-DIO3印记域miRNA簇/c-Met在芹菜素逆转膀胱癌顺铂耐药的作用及机制研究

Bladder cancer is the most common urological malignancy in our country. Cisplatin resistance is a large difficulty in the treatment of bladder cancer. Thus to explore the molecular mechanism of cisplatin resistance in bladder cancer and to seek a new way of treatment are of great significance. We found in previous studies, a natural element of flavonoids apigenin can reverse the cisplatin resistance in bladder cancer, while circ_0001658, DLK1-DIO3 miRNA cluster, c-Met and its downstream pathways also changed significantly at the same time. Therefore, we speculate that circ_0001658/DLK1-DIO3 miRNA cluster/c-met axis plays an important role in the form of cisplatin resistance and reversal of cisplatin resistance caused by apigenin in bladder cancer. This project plans to clarify the role and its underlying mechanism of circ_0001658/DLK1-DIO3 miRNA cluster/c-met axis in cisplatin resistance of bladder cancer and the reversal effect by apigenin by building a drug-resistant cell line, circRNA sequencing, bioinformatics tools, biological function experiment, RNA FISH, animal experiments, etc. The successful completion of this project will complement the mechanism of cisplatin resistance in bladder cancer, and provide a new strategy for targeted therapy and chemotherapy for drug resistant tumors.

膀胱癌是我国最常见的泌尿系统恶性肿瘤，顺铂化疗耐药是膀胱癌治疗的难点和重点，探索膀胱癌顺铂耐药发生的分子机制并寻求新的诊断和治疗方式意义重大。我们在前期研究中发现一种天然的黄酮类化合物芹菜素可以逆转膀胱癌顺铂耐药，同时环状RNA circ_0001658、DLK1-DIO3印记域miRNAs及c-Met和下游通路也相应变化，因此推测circ_0001658/DLK1-DIO3印记域miRNA簇/c-Met轴在膀胱癌顺铂耐药的形成和芹菜素逆转顺铂耐药过程中发挥重要作用。本项目拟通过构建耐药细胞株、circRNA芯片、生物信息学、生物功能实验、RNA FISH、尾静脉注射肺转移模型等技术，阐明circ_0001658/DLK1-DIO3印记域miRNA簇/c-Met在膀胱癌顺铂耐药和芹菜素作用中的具体机制。本项目的成功实施将为膀胱癌顺铂耐药的机制作一补充，为耐药肿瘤的诊断和化疗提供新的策略。

膀胱癌是我国最常见的泌尿系统恶性肿瘤，顺铂化疗耐药是膀胱癌治疗的难点和重点，探 索膀胱癌顺铂耐药发生的分子机制并寻求新的诊断和治疗方式意义重大。我们发现一种天然的黄酮类化合物芹菜素可以逆转膀胱癌顺铂耐药，同时环状RNA circ_0001658、DLK1-DIO3印记域miRNAs及c-Met和下游通路也相应变化，因此推测circ_0001658/DLK1-DIO3印记域miRNA簇/c-Met轴在膀胱癌顺铂耐药的形成和芹菜素逆转顺铂耐药过程中发挥重要作用。研究通过构建耐药细胞株、circRNA芯片、生物信息学、生物功能实验、RNA FISH、尾静脉注射肺转移模型等技术，明确了芹菜素对UM-UC3/DDP细胞顺铂敏感性的影响及抑癌作用，构建了膀胱癌顺铂耐药细胞株，检测耐药特性。在体外细胞实验中，将芹菜素与顺铂单独或联合作用于UM-UC3/DDP细胞后检测细胞的耐药水平和增殖能力，凋亡和细胞周期，细胞侵袭和迁移能力。进一步在体内实验验证，建立NOD-SCID小鼠皮下成瘤模型与经尾静脉注射的肺转移模型，腹腔注射顺铂和芹菜素进行模拟临床化疗。我们筛选出明显上调的DLK1-DIO3印记域miRNAs，验证DLK1-DIO3印记域miRNAs在芹菜素调控细胞耐药中的作用，在耐药细胞中过表达上述miRNAs，检测细胞的耐药水平、细胞增殖、凋亡、侵袭与迁移等生物学功能。在耐药细胞中过表达上述miRNAs，通过qRT-PCR和Western blot检测c-Met的变化。利用荧光素酶双报告系统证实上述miRNAs对c-Met的直接调控。研究明确circ_0001658介导膀胱癌顺铂耐药的生物学功能，揭示其分子机制。研究成果阐明circ_0001658/DLK1-DIO3印记域miRNA簇/c-Met在膀胱癌顺铂耐药和芹菜素作用中的具体机制，为膀胱癌顺铂耐药的机制作一补充，为耐药肿瘤的诊断和化疗提供新的策略。