膀胱癌化疗耐药相关基因MED7的筛选和作用机制研究

Drug resistance is one of the main causes of chemotherapy failure and recurrence of bladder cancer. The study of drug resistance mechanism is of great significance to enhance the clinical effect of bladder cancer. Combined with the genome data and clinical information, our group has constructed gene bank to screen resistant genes of bladder cancer by the CRISPR system. Among those candidate genes, MED7 gene was correlated with pathological staging, overall prognosis, and prognosis after chemotherapy by clinical analysis. Furthermore, knockout of MED7 gene could lead to abnormal activation of TGF-β resistance pathway. Therefore, we speculated that as MED7 participated in the process of chemo-resistance by TGF-β pathway or other mechanisms. Here, 1) we intend to study the physiological role of MED7 in drug resistance through cell experiments, animal model and clinical pathological specimens. 2) Furthermore, we will knock out the key genes of TGF-β pathway in MED7 knockout bladder cancer cells, to explore the downstream molecules regulated by MED7 and the resistance mechanism. 3) Finally, we will perform transcriptome analysis of MED7 knockout bladder cancer cells treated with drugs, and explore other possible mechanisms of MED7 in drug resistance by the CRISPR technique. This study will provide not only important molecular markers for drug resistance of bladder cancer, but also scientific fundamental for the personalized medicine.

耐药是膀胱癌化疗失败和复发主要原因之一，耐药机制研究对提升膀胱癌临床效果具有重要意义。项目组利用膀胱癌基因组数据并结合临床信息建立化疗耐药候选基因库，通过 CRISPR系统和药物实验筛选得到MED7基因，进一步临床分析表明 MED7与膀胱癌病理分期、患者总预后和化疗后预后等相关，MED7基因敲除可导致TGF-β耐药通路异常激活。推测MED7作为膀胱癌耐药相关基因，通过TGF-β通路参与了化疗耐药过程。本项目拟：1）细胞体外实验、动物模型和临床标本研究MED7参与和影响膀胱癌化疗耐药的作用；2）敲除MED7缺失膀胱癌细胞中TGF-β通路关键基因以探索MED7功能关联分子及影响化疗耐药过程的作用机制；3）药物处理MED7缺失膀胱癌细胞后转录组分析并结合CRISPR技术探索MED7参与耐药过程其它可能新机制。本课题将为膀胱癌化疗耐药研究提供重要分子标志物并为临床个体化用药策略制定提供科学依据。

膀胱癌是泌尿系统最常见的十大肿瘤之一，其中男性发病率远高于女性，是泌尿肿瘤领域面临的重要难题。目前手术治疗联合化疗、放疗等手段虽起到一定的遏制效果，但化疗耐药性的产生严重制约了临床用药效果。因此，膀胱癌化疗耐药是当前迫切需要解决的临床难题，寻找有效的耐药靶点并解析其作用机制将为膀胱癌的临床治疗提供理论基础。我们前期通过CRISPR系统高通量筛选膀胱癌耐药候选基因，构建了膀胱癌耐药候选基因库及相应的gRNA文库﹑Cas9膀胱癌细胞系及稳定表达的基因敲除细胞系，确定MED7为膀胱癌的耐药相关基因。进一步通过细胞实验及动物实验对MED7在膀胱癌中的调控作用进行了深入探讨，发现MED7为膀胱癌的有效调控因子，并且能够增敏膀胱癌一线化疗药物。最后通过细胞测序分析及细胞功能实验验证，初步明确MED7可通过激活ROS相关的内质网应激途径促进膀胱癌细胞凋亡﹑增敏膀胱癌一线化疗药物顺铂，吉西他滨的治疗效果。