Ref-1介导的MAPK信号通路参与卵巢癌铂类耐药的机制研究

Epithelial ovarian cancer is a disease with high incidence and mortality. The major problem is platinum-resistance in ovrian cancer clinic therapy. Our former research found that the level of Ref-1 expression were upregulated by cisplatin, Ref-1 siRNA enhanced apoptosis induced by cisplatin in ovarian cancer cells. Meanwhile cisplatin stimulated p38 phosphorylation and apoptosis was attenuated by Ref-1 overexpression. We hypothesized that the Ref-1 gene could neutralize apoptosis induced by cisplatin via the MAPK sigaling pathway in ovarian cancer. Based on our previous study, we would use the techniques of RT-PCR,western blot, MTT, TUNEL etc. to detect the relationship between Ref-1、MAPK pathway and apoptosis induced by cisplatin in ovarian cancer cells. We would construct Ref-1 recombinant plasmid、Ref-1 Cys65 mutant plasmid and Ref-1 siRNA/Ref-1 shRNA, and identify the impact of Ref-1 redox function on the AMPK signaling pathway and the apoptosis induced by cisplatin of ovarian cancer cells in vivo and vitro. Finally, we would explore the regulation mechanisms of Ref-1 on MPAK signaling pathway in apoptosis induced by cisplatin through detection of the MAPK poteins and the targetd aopoptosis proteins of MAPK pathway in ovarian cancer. Our study would help clarify the regulated mechanism of Ref-1 on MAPK signaling pathway in apoptosis induced by cisplatin, and provide a theoretical basis for the use of Ref-1 and MAPK signaling pathway to achieve molecular targeted therapy of ovarian cancer.

卵巢癌是常见恶性肿瘤，铂类耐药是临床面临主要问题。前期发现①顺铂诱导卵巢癌细胞Ref-1表达升高，Ref-1 siRNA增加顺铂诱导的细胞凋亡；②顺铂诱导卵巢癌细胞p38磷酸化，Ref-1过表达降低顺铂诱导的p38磷酸化并减少细胞凋亡。结果提示Ref-1介导MAPK信号通路参与调控顺铂诱导的细胞凋亡。本课题拟通过RT-PCR、western blot、TUNEL、FCM等技术观察顺铂诱导凋亡与Ref-1、MAPK信号通路的关系；构建Ref-1质粒、Ref-1Cys65突变质粒和Ref-1 siRNA，从体内和体外实验观察Ref-1对MAPK信号通路及顺铂诱导凋亡的影响；通过检测MAPK通路相关分子表达及活性和下游凋亡蛋白表达探讨Ref-1介导MAPK通路对顺铂诱导凋亡的调控机制。本课题将阐明Ref-1介导的MAPK通路在肿瘤细胞对铂类药物应激中的作用机制，为肿瘤细胞铂类耐药机制提供新依据。

卵巢癌是常见恶性肿瘤，铂类耐药是临床面临主要问题。本课题在前期工作基础上发现：（1）卵巢癌细胞顺铂耐药性与ROS水平、MAPK通路活化相关；（2）体内试验和体外实验均证实Ref-1过表达可显著提高卵巢癌细胞增殖、迁移和侵袭能力，降低卵巢癌细胞对铂类的敏感性，其中Ref-1氧化还原功能对于促进细胞增殖、侵袭迁移、铂类耐药形成过程中发挥主导作用；（3）mRNA芯片、microRNA芯片筛选获得Ref-1WT组、Ref-1Cys65组之间的差异基因，根据芯片筛选结果在卵巢癌细胞中初步验证Ref-1通过氧化还原调控RAP1A/MAPK/ERK信号通路，从而参与了卵巢癌细胞铂类耐药性的形成；（4）miRNA芯片结果显示与Ref-1氧化还原功能相关的miRNA靶基因主要富集在肿瘤通路、细胞骨架调控、MAPK通路、粘附连接、ECM受体通路等方面。