泛素化降解ARHGAP10致卵巢癌顺铂耐药的分子机制研究

Platinum-based drugs resistance directly relates to the survival of ovarian cancer patients, so it is a clinical problem to be solved urgently to reveal the resistance mechanism in ovarian cancer. Our previous study has confirmed that ARHGAP10 is depressed in ovarian cancer and is associated with poor prognosis, which may involve in cisplatin resistance in ovarian cancer. Here, we focus on the following three aspects: (1) SKOV3/DDP and OVCAR3/DDP resistant cell lines were selected to overexpress / silencing WWP2 gene to determine the effect of WWP2 on ARHGAP10 ubiquitination by Co-IP combining with mass spectrometry. (2) Laser scanning confocal microscopy and classical molecular biology experiments are carried out to detect the effect of ARHGAP10 on the autophagy in ovarian cancer cell lines. (3) To analyze the expression of ARHGAP10, WWP2 and autophagy-related molecules in cisplatin-resistant and cisplatin-sensitive tissues, a large number of clinical ovarian cancer samples were collected to establish the correlation between the related molecules and cisplatin resistance in ovarian cancer. This research will reveal the molecular mechanism of ARHGAP10-mediated cisplatin resistance in post-transcription and autophagy regulation levels, and will provide evidence for clinical judgment of cisplatin resistance in ovarian cancer.

铂类药物耐药直接关联卵巢癌患者生存期，揭示卵巢癌耐药机制，解决耐药问题是临床亟待解决的难题。课题组前期研究已证实ARHGAP10在卵巢癌中低表达并与预后不良相关；而且可能涉及卵巢癌顺铂耐药机制。本课题重点研究以下三个环节：（1）选择SKOV3/DDP和OVCAR3/DDP耐药细胞株，干扰/过表达WWP2基因，采用Co-IP结合质谱技术观察WWP2对ARHGAP10泛素化的影响。（2）采用激光共聚焦及经典分子生物学方法检测细胞自噬现象及相关分子表达，观察ARHGAP10对卵巢癌耐药细胞株自噬的影响。（3）卵巢癌临床大样本对照分析顺铂耐药与顺铂敏感组织样本，验证ARHGAP10、WWP2及自噬相关分子表达情况，并建立相关分子与卵巢癌顺铂耐药的关联。本课题研究在细胞转录后水平及自噬调控环节揭示ARHGAP10介导的卵巢癌耐药分子机制，并为临床判断卵巢癌顺铂耐药提供依据。

一、项目背景：.铂类药物耐药直接关联卵巢癌患者生存期，揭示卵巢癌耐药机制，解决耐药问题是临床亟待解决的难题。课题组前期研究已证实ARHGAP10在卵巢癌中低表达并与预后不良相关；而且可能涉及卵巢癌顺铂耐药机制。..二、主要研究内容：.（1）选择SKOV3/DDP和OVCAR3/DDP耐药细胞株，干扰/过表达NEDD4基因，采用Co-IP结合质谱技术观察NEDD4对ARHGAP10泛素化的影响。（2）采用激光共聚焦及经典分子生物学方法检测细胞自噬现象及相关分子表达，观察ARHGAP10对卵巢癌耐药细胞株自噬的影响。（3）卵巢癌临床大样本对照分析顺铂耐药与顺铂敏感组织样本，验证ARHGAP10、NEDD4及自噬相关分子表达情况，并建立相关分子与卵巢癌顺铂耐药的关联。..三、重要结果：.1、ARHGAP10在顺铂耐药卵巢癌组织/细胞中的表达显著低于顺铂敏感卵巢癌组织/细胞；.2、ARHGAP10通过抑制自噬增强卵巢癌细胞对顺铂的敏感性；.3、NEDD4可能通过泛素化降解ARHGAP10的表达，进而促进细胞自噬抑制卵巢癌细胞的凋亡；..四、关键数据：.1、稳定上调卵巢癌顺铂耐药细胞系SKOV3/DDP细胞ARHGAP10表达能增强SKOV3/DDP细胞对顺铂的敏感性，促进顺铂诱导的细胞凋亡；ARHGAP10过表达后，SKOV3/DDP细胞中LC3II表达降低、Caspase3和PARP的表达升高；自噬通路相关标记物p-AMPK、ULK1及p-ULK1表达降低、AMPK的表达无明显变化；而添加自噬激动剂AICAR则能抑制ARHGAP10的上述生物学功能。.2、通过人类泛素连接酶-底物相互作用预测系统数据库预测、PCR检测验证并进一步采用免疫共沉淀实验证实ARHGAP10 与NEDD4 存在相互作用；采用慢病毒干扰载体稳定下调卵巢癌SKOV3/DDP细胞株NEDD4表达：Western blot检测提示ARHGAP10的表达升高。.3、采用慢病毒干扰载体稳定下调卵巢癌SKOV3/DDP细胞株NEDD4表达能增强顺铂诱导的细胞凋亡。..五、科学意义：.本课题验证了ARHGAP10对卵巢癌细胞顺铂耐药的调控作用，以及调控相关分子机制。结果提示ARHGAP10在卵巢癌组织中是一个重要的调控卵巢癌顺铂敏感性的基因，是一个潜在的卵巢癌治疗靶点，研究结果具有一定的临床转化价值。