双功能酶APE1在卵巢癌干细胞化疗耐药中的作用及分子机制研究

Chemotherapy-resistant is the most important reason of ovarian cancer recurrence and metastasis, and ovarian cancer stem cells play an important role in chemotherapy resistance. With the help of fluorescence-activated flow stem cell separation technology, our group used CD133/CXCR4 as markers, obtained the ovarian cancer stem cell subsets which possess tumorigenicity, self-proliferation, colony formation characteristics from ovarian cancer cell lines, and confirmed them to be associated with ovarian cancer platinum sensitivity. The preliminary study also found that the multifunctional enzyme APE1 is related to the occurrence, development and prognosis of ovarian cancer and participate in the formation of ovarian cancer platinum-resistant. However, the influence of APE1 on ovarian cancer stem cell biology characteristics and the mechanism of APE1 participating in platinum resistance of ovarian cancer have not been reported. This research is proposed on the basis of preliminary work. We are going to detect the expression and localization of APE1 in ovarian cancer tissue samples by means of immunohistochemical methods; construct the APE1 different dominant negative mutant and downregulate APE1 expression using site-directed mutagenesis, RNA interference and other technologies , observe its influence on the biological characteristics of ovarian cancer stem cells and platinum sensitivity in vivo and vitro; explore known and unknown pathway of APE1 participating in chemotherapy resistance of ovarian cancer, evaluate its significance as an ovarian cancer gene therapeutic target, and clarify the mechanism of resistance to chemotherapy from the molecular level.

化疗耐药是卵巢癌复发转移的主要原因，卵巢癌干细胞在其中发挥着重要作用。我们课题组借助荧光激活的流式干细胞分选技术，采用CD133/CXCR4作为标志物，成功地从卵巢癌细胞系中得到了具有成瘤性、自增殖、克隆形成等特性的卵巢癌干细胞亚群，证实其与卵巢癌铂类敏感性相关。前期研究还发现，双功能酶APE1与卵巢癌发生发展及预后有关并参与卵巢癌铂类耐药形成。但APE1对卵巢癌干细胞生物学特性的影响及参与铂类耐药的机制仍有待于进一步研究。本课题拟在前期工作基础上，通过免疫组化等方法大样本检测APE1在卵巢癌组织中的表达及定位；利用定点突变、RNA干扰等技术构建APE1不同功能显性负突变体及下调APE1表达水平，从体内、外观察其对卵巢癌干细胞生物学特性及铂类敏感性的影响；探讨APE1参与卵巢癌化疗耐药的已知通路及未知通路，评价其作为卵巢癌基因治疗靶点的意义，从分子水平阐明APE1参与卵巢癌化疗耐药的机制。

化疗耐药是卵巢癌复发转移的主要原因，寻求有效的化疗耐药逆转策略是提高卵巢癌疗效的关键。我们课题组借助荧光激活的流式干细胞分选技术，采用CD133/CXCR4作为标志物，成功地从卵巢癌细胞系中得到了具有成瘤性、自增殖、克隆形成等特性的卵巢癌干细胞亚群，证实其与卵巢癌铂类敏感性相关。本课题在前期工作基础上，通过免疫组化检测127例上皮性卵巢癌组织中APE1的表达，统计结果表明APE1的表达差异与病理类型、FIGO分期、分化程度及铂类耐药有关；体内实验和体外实验均证实，APE1高表达可显著促进卵巢癌细胞增殖和侵袭能力，并降低卵巢癌细胞顺铂敏感性，其中APE1的氧化还原功能发挥主要作用。进一步成功构建Redox-APE1 siRNA的慢病毒，能够显著抑制卵巢癌细胞增殖，导致G1/S期阻滞，并增加其对顺铂的敏感性。进一步研究发现Redox-APE1参与卵巢癌化疗耐药的相关分子和通路，评价其作为卵巢癌基因治疗靶点的意义，从分子水平阐明APE1的氧化还原功能参与卵巢癌化疗耐药的机制，可能为：1）Redox-APE1高表达能够通过上调多药耐药相关基因（ABCG2、MDR1、MRP2、MRP4、MRP5及MRP6）表达水平，下调TOPOⅡ水平，从而降低卵巢癌细胞对顺铂的敏感性，参与调控多药耐药机制；2）同时Redox-APE1高表达能够降低Bcl-2基因抗凋亡作用，影响Bax促凋亡作用，进而抑制由顺铂诱导的卵巢癌细胞发生凋亡；3）高表达的Redox-APE1能够显著上调IL-6水平，但却几乎不改变STAT3 mRNA水平，而下调APE1的表达却能够显著抑制IL-6和STAT3水平，降低细胞上清中IL-6的含量；4）双荧光素酶报告基因检测结果表明Redox-APE1的水平与STAT3启动子转录活性呈正相关。给予IL-6刺激后高表达Redox-APE1能够显著上调STAT3启动子相对活性，表明Redox-APE1在APE1调控STAT3启动子活性中发挥重要作用。这些结果为其作为基因治疗靶点提供有效依据。利用基因芯片技术，我们还发现APE1调控的信号通路主要涉及MAPK信号通路、细胞粘附、细胞吞噬、阿米巴运动等方面，功能网络信号通路分析结果表明， APE1与TP53、HIF1α、PCNA等基因存在调控关系。