【作者】 李佳；

【导师】 辛晓燕； 张伟；

【作者基本信息】 第四军医大学 ， 妇产科学， 2013， 博士

【摘要】 卵巢癌是严重威胁女性健康的恶性肿瘤之一，其恶性程度在女性生殖系统肿瘤中占第1位。由于缺乏可靠的早期检测方法，约70%的患者初次诊断时已经为III或IV期，并且出现广泛转移，严重影响疾病的治疗效果，降低患者生存质量。目前国内外对卵巢癌初诊患者采用的标准治疗方案是联合积极彻底的肿瘤细胞减灭术和以铂类为基础的化疗治疗。根治性手术和化疗方案的进步发展使得卵巢癌的治疗有所改善。但是，肿瘤细胞原发性或继发性的化疗耐药是影响化疗效果，造成肿瘤复发、转移及死亡率居高不下的重要原因，给临床治疗带来极大困扰。因此，研究卵巢癌复发和化疗耐药的机制并采取有效的预防和治疗措施已成为卵巢癌治疗研究的当务之急。脱嘌呤/脱嘧啶核酸内切酶（Apurinic/apyrimidinic endonuclease，APE1）是DNA碱基切除修复（Base excision repair，BER）途径中的一种多功能蛋白酶，不仅具有核酸内切酶活性，在DNABER途径中发挥关键限速酶的作用，而且还能通过其氧化还原功能调控多种重要转录因子的活性，参与肿瘤的发生发展、氧化应激、细胞周期调控及凋亡等多种关键的细胞反应。APE1在DNA损伤修复和凋亡通路调控两大途径中均发挥重要作用。多项研究结果均表明，APE1的表达不仅与多种肿瘤的发生发展和预后相关，而且与多种肿瘤的放化疗敏感性相关。鉴于APE1上述的功能特点，我们推测APE1在肿瘤细胞化疗耐药中扮演着重要角色，可能成为极具潜力的肿瘤治疗靶点。我们前期发现①APE1的表达和细胞定位与卵巢癌发生发展、铂类化疗敏感性及预后相关；②卵巢癌顺铂耐药株A2780/cis中APE1的表达水平显著高于卵巢癌顺铂敏感株A2780；③高表达APE1能显著降低卵巢癌细胞对顺铂的敏感性。综合前期的研究基础，我们提出并证实了APE1在介导卵巢癌铂类耐药中发挥着重要作用。近年来多项研究证实，APE1的DNA修复功能在多种肿瘤的发生发展、化疗耐药及预后中扮演着重要角色。APE1具有3’-5’核酸外切酶作用，在错配的脱氧核糖核苷类似物的切除中发挥作用，抑制这种作用能够增加吉西他滨类化疗药物的治疗作用。体内体外实验均表明，通过RNA干扰或特异性小分子抑制剂抑制APE1的DNA修复功能，并与放化疗联合应用，在一定程度上能够增强放化疗对肿瘤细胞的杀伤能力。尽管针对APE1在卵巢癌铂类耐药过程中发挥的作用取得了一些研究结果，但仍有很多问题亟待深入探讨：我们对其DNA修复功能已有充分的了解，但对其氧化还原功能却知之甚少。APE1的氧化还原功能在其参与肿瘤发生发展、氧化应激、细胞周期调控及凋亡等多种关键细胞反应中发挥着重要作用，那么APE1的氧化还原功能在卵巢癌铂类耐药形成中又扮演怎样的角色？干预APE1是否能有效逆转卵巢癌铂类耐药呢？为此，我们在前期研究的基础上，进行了下列研究工作：根据APE1结构功能特点，构建含有APE1基因及其氧化还原区的重组腺病毒Ad5-APE1-EGFP和Ad5-Redox-APE1-EGFP；将Ad5-APE1-EGFP和Ad5-Redox-APE1-EGFP重组腺病毒分别感染卵巢癌细胞，荧光显微镜下观察绿色荧光蛋白（EGFP）的表达检测感染情况，qRT-PCR和Western blot分别检测重组腺病毒感染后卵巢癌细胞中APE1mRNA和蛋白水平变化； MTT、平板克隆形成实验及侵袭实验检测感染APE1不同重组腺病毒后卵巢癌细胞增殖能力及侵袭能力的变化；药物敏感性实验检测感染APE1不同重组腺病毒，对卵巢癌细胞顺铂敏感性的影响；流式细胞术检测感染APE1不同重组腺病毒后卵巢癌细胞周期的变化； Hoechst33258染色法检测APE1不同重组腺病毒感染后，顺铂诱导的卵巢癌细胞凋亡的变化；激光共聚焦检测APE1不同重组腺病毒感染后卵巢癌细胞中APE1的亚细胞定位；体内实验建立卵巢癌裸鼠皮下荷瘤模型，进一步观察APE1及其氧化还原功能在卵巢癌化疗耐药中的重要作用及其对肿瘤增殖的影响；构建针对APE1氧化还原区的Redox-APE1siRNA慢病毒下调Redox-APE1表达后，通过MTT法、药物敏感性实验及流式细胞术检测细胞周期观察其对卵巢癌细胞生物学行为的影响； qRT-PCR检测改变APE1表达水平对耐药相关基因的影响；⑴qRT-PCR和Westernblot法检测改变APE1表达水平对凋亡相关基因的影响；⑵qRT-PCR检测改变APE1表达水平对Jak/STAT通路基因表达的影响；⑶在卵巢癌A2780细胞中上调和下调APE1的表达，利用双荧光素酶报告基因系统检测了APE1表达水平对STAT3启动子活性的影响。通过以上实验我们获得了下列结果：成功构建含有APE1基因及其氧化还原区的重组腺病毒Ad5-APE1-EGFP和Ad5-Redox-APE1-EGFP；将Ad5-APE1-EGFP和Ad5-Redox-APE1-EGFP重组腺病毒分别感染卵巢癌细胞后，荧光显微镜下可观察到95%以上细胞能够表达EGFP，qRT-PCR和Western blot分别表明重组腺病毒感染后卵巢癌细胞中APE1mRNA和蛋白水平均显著升高（P＜0.05）； MTT、平板克隆形成实验及侵袭实验表明，感染APE1不同重组腺病毒后能够促进卵巢癌细胞的增殖，显著增强卵巢癌细胞的侵袭能力（P＜0.05）；药物敏感性实验证实感染APE1不同重组腺病毒后，卵巢癌细胞对顺铂的敏感性显著下降（P＜0.05）；流式细胞术结果表明，感染APE1不同重组腺病毒后，卵巢癌细胞周期G0/G1期比例下降，S期比例增加； Hoechst33258染色法显示，APE1不同重组腺病毒感染后，顺铂诱导的卵巢癌细胞凋亡发生减少；激光共聚焦检测结果显示，Redox-APE1重组腺病毒感染后卵巢癌细胞中APE1表达主要定位于细胞核，而APE1重组腺病毒感染后卵巢癌细胞中APE1表达主要表现为细胞核或细胞浆/细胞核共表达；成功建立卵巢癌裸鼠皮下荷瘤模型，通过体内实验进一步验证APE1及其氧化还原功能在卵巢癌化疗耐药中的重要作用及其对肿瘤增殖的影响；成功构建Redox-APE1siRNA慢病毒下调Redox-APE1表达后，能够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（P＜0.05）； Real-time PCR结果表明在卵巢癌A2780细胞和A2780/cis细胞中高表达的APE1及Redox-APE1通过上调多药耐药基因MDR1、MRP4及ABCG2的mRNA水平，下调TOPOⅡmRNA水平；而在卵巢癌SKOV3细胞和SKOV3/cis细胞中，高表达全长APE1及Redox-APE1在显著上调MDR1同时，能够明显增加MRP2、MRP5及MRP6的mRNA水平，从而参与调控多药耐药机制；⑴APE1及Redox-APE1的高表达能够调控Bcl-2基因发挥抗凋亡作用，影响Bax发挥促凋亡作用，进而抑制由顺铂诱导的卵巢癌细胞发生凋亡；⑵高表达APE1及Redox-APE1能够显著上调IL-6mRNA水平和细胞上清中IL-6的含量，但却几乎不改变STAT3mRNA水平，而下调APE1的表达却能够显著抑制IL-6和STAT3mRNA水平，降低细胞上清中IL-6的含量；⑶双荧光素酶报告基因检测结果表明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APE1及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。综上所述，本课题在前期工作基础上成功构建含有APE1基因及其氧化还原区的重组腺病毒及下调Redox-APE1表达水平的Redox-APE1siRNA慢病毒，从细胞水平和动物水平分别观察其对卵巢癌细胞生物学特性及铂类敏感性的影响，进一步为APE1的氧化还原功能参与卵巢癌铂类耐药机制提供直接证据，也为深入研究以APE1为逆转卵巢癌铂类耐药的治疗靶点提供新的思路。更多还原

【Abstract】 Epithelial ovarian cancer (EOC), is one of the most frequent malignancies in femalereproductive system and has the highest mortality rate of all gynecological cancersworldwide. This high mortality is attributed in part to the lack of any reliable earlydetection method resulting in the majority of patients being diagnosed with advancedstage III or stage IV disease. Currently, the standard of care for ovarian cancer isaggressive surgical debulking followed by platinum-based combination chemotherapy.Although ovarian cancer is among the most chemosensitive malignancies at the time ofinitial treatment(surgery and taxane/platinum-based chemotherapy), most patients willultimately develop tumor recurrence and succumb to chemoresistant disease. Primary orsecondary chemoresistance is an important factor affecting chemotherapy and causedtumor recurrence, metastasis and higher mortality, which bring huge difficulties to clinicaltreatment. Therefore, it’s very importment to clarify the mechanisms of ovarian cancerrecurrence and chemoresistance and to take some effective measures to prevention and treatment of ovarian cancer.APE1is a multifunctional protein in DNA base excision repair pathway. It not onlyhas the activity of endonuclease but also is an essential enzyme in base excision repairpathway. It also acts as a major redox-signaling factor that involve in transcription factorregulation, tumor progression, oxidative stress, cell cycle and apoptosis. APE1plays animportment role in DNA damage and repair and apoptosis pathway regulation. A growingbody of evidence has demonstrated that APE1is not only critical for tumorigenesis andprogression in several human tumors and cancer cell lines, but also closely related to thechemosensitivity and radiosensitivity. It is reasonable to postulate that APE1maycontribute to the molecular mechanism of resistance to cisplatin-based chemotherapy, as avery potential tumor therapeutic target.Our previous results find that:①The expression and subcellular localization ofAPE1are involved in tumorigenesis, progression, chemosensitivity and prognosis inovarian cancer.②The cisplatin-resistant A2780/cis cells demonstrated higher expressionprotein levels of APE1than the cisplatin-sensitive A2780cells.③The overexpression ofAPE1had significant enhancement in cisplatin sensitivity. Above all the data, weproposed and confirmed that APE1involved in this resistance may provide new treatmentmodalities for ovarian cancer.A growing body of evidence has demonstrated that repair functions of APE1plays animportment role in tumorigenesis, progression, chemosensitivity and prognosis of variouscancers. Selective targeting of this DNA repair enzyme using RNA interference or specificsmall-molecule inhibitors blocking APE1repair functions have been shown to be effectiveinsensitizing cancer cells to both adiation and chemotherapy in vivo and in vitro.Some results about APE1’s role in the chemoresistance process in ovarian cancerhave been clarified. However, a number of questions are unclear. Our understanding ofAPE1DNA repair functions have been fully, but little is known about its Redox function.What important role does the redox function of APE1have in the process ofcisplatin-based chemotherapy? Whether intervention APE1can effectively enhancechemosensitivity in ovarian cancer? On the basis of the results of preliminary studies, some works have been done：According to characteristics of APE1structure, we constructed Ad5-APE1-EGFP andAd5-Redox-APE1-EGFP recombinant adenovirus, which contains APE1gene and itsredox structure, respectively. To detect the effect of the overexpression of APE1and itsredox in ovarian cancer cells, The infection efficiency was confirmed byfluorescence microscope and qRT-PCR and Western blot were performed to evaluate theexpression of APE1mRNA and protein respectively. MTT assays, clone formation assaysand transwell assays were performed to evaluate the effects of Ad5-APE1-EGFP andAd5-Redox-APE1-EGFP recombinant adenovirus on cell proliferation and Invasion. Drugsensitivity assays were performed to determine the cisplatin sensitivity of ovarian cancercells. The effect of Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFP recom-binant adenovirus on cell cycle was observed by flow cytometry analysis in infectedovarian cancer cells. Hoechst33258staining were performed to showed the apoptosis ratein ovarian cancer cells infected with Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFPrecombinant adenovirus after treated with cisplatin.Immunofluorescence was used toobserves ubcellular localization of APE1in ovarian cancer cells infected with Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFP. We constructed subcutaneous tumor model in nudemice and further confirmed that APE1and its resox played an iomportment role inchemoresistance in ovarian cancer and in tumor proliferation in vivo. We also constructedRedox-APE1siRNA lentivirus to down regulation the expression of APE1, and observedthe impact on biological function in ovarian cancer cell lines.Some results have been obtained：According to characteristics of APE1structure, wesuccessfully constructed recombinant adenovirus Ad5-APE1-EGFP andAd5-Redox-APE1-EGFP, Which contains APE1gene and its redox structure, respectively.The95%of ovarian cancer cells were infected with Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFP recombinant adenovirus by fluorescence microscope and qRT-PCR andWestern blot results showed that the expression of APE1mRNA and protein weresignificantly increased in ovarian cancer cells infected with Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFP, respectively（P＜0.05）. MTT assays, clone formation assays and transwell assays indicated that ovarian cancer cells infected with Ad5-APE1-EGFP andAd5-Redox-APE1-EGFP can increase the cell proliferative activity and the invasionability. Drug sensitivity assays demonstrated that the chemosensitivity of cisplatin-basedchemotherapy was significantly reduced. Cell cycle analysis by flow cytometry showedthat ovarian cancer cells infected with Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFPresulted in accumulation in G2and S phase. While the proportion of G0/G1phase weredecreased. Hoechst33258staining showed that the apoptosis rate was weakly decreased inovarian cancer cells infected with Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFP aftertreated with cisplatin. Immunofluorescence detection showed that the subcellular locationof APE1in ovarian cancer cells were cytoplasmic. We further confirmed that APE1andits resox played an iomportment role in chemoresistance in ovarian cancer and in tumorproliferation in vivo. We also successfully constructed Redox-APE1siRNA lentivirus todown regulation the expression of Redox-APE1, and observed the impact onbiological function in ovarian cancer cell lines. These results provided strong evidencesfor highlighting the potential of APE1to serve as a target for cancer therapeutics.In conclusion, on the basis of preliminary work, we successfully constructedrecombinant adenovirus Ad5-APE1-EGFP and Ad5-Redox-APE1-EGFP, Which containsAPE1gene and its redox structure, respectively. We also successfully constructed APE1siRNA lentivirus to down regulation the expression of APE1. We observed the impact onbiological function and the chemosensitivity to cisplatin in vivo and in vitro. These resultsfurther provided strong evidences for the role of APE1in chemoresistance of ovariancancer and also may provide new treatment modalities for ovarian cancer.更多还原