【作者】 何春容；

【导师】 钟梅；

【作者基本信息】 南方医科大学 ， 妇产科学， 2010， 博士

【摘要】 研究背景卵巢癌是女性生殖系统三大恶性肿瘤之一,其恶性程度高,易发生血行、淋巴转移和局部浸润播散,易对化疗药物产生耐药,因此预后较差。锌指蛋白217(ZNF217)基因位于人类染色体20q13.2,是20号染色体上发现的新癌基因,它编码的Krupple样转录因子属于锌指蛋白家族。ZNF217参与了包括卵巢癌、乳腺癌、肺癌、胃肠道癌等多种肿瘤的发生发展过程。本课题组前期发现ZNF217基因扩增程度与卵巢癌的临床分期有关,对卵巢癌细胞进行ZNF217基因的RNA干扰后,癌细胞的运动、侵袭能力明显降低,提示ZNF217与卵巢癌的侵袭转移能力有相关性。国外学者对乳腺癌的研究发现,ZNF217能削弱化疗药物阿霉素对癌细胞凋亡的诱导作用,通过RNA干扰抑制ZNF217基因表达,能改善卵巢癌细胞对阿霉素的耐药性。但目前尚无关于ZNF217与卵巢癌化疗耐药的关系的研究。对化疗药物产生耐药是肿瘤治疗失败的一个重要原因,而肿瘤出现化疗耐药的重要机制之一为凋亡调控基因所介导的凋亡通路的阻断。凋亡抑制蛋白(IAPs)是一类独特的抗凋亡蛋白家族,已发现8个人类IAPs家族成员：XIAP、survivin、c-IAP1、和livin等。多数IAPs能直接抑制凋亡终末效应酶caspase-3、caspase-7及caspase-9的活性,从而发挥抗凋亡作用。IAPs通过阻断凋亡,促进肿瘤对化疗药物产生耐药性,其过度表达与肿瘤治疗失败有关。在卵巢癌组织中,XIAP和survivin表达均增强,二者过表达可抑制卵巢癌细胞的凋亡,与卵巢癌细胞对顺铂和紫杉醇耐药有关。将正义XIAP cDNA转染顺铂敏感卵巢癌细胞株后,发现XIAP过表达可诱导存活因子Akt磷酸化使其失活,并阻断顺铂诱导的细胞凋亡。有研究认为卵巢癌细胞对顺铂产生耐药可能与XIAP抑制caspase-3的激活有关。临床资料分析显示,survivin的表达与紫杉醇耐药相关,将survivin cDNA稳定转染入不表达survivin基因的人卵巢癌细胞株后,癌细胞对紫杉醇的耐药性提高了4-6倍,同时紫杉醇诱导的细胞凋亡率明显降低。有人用低浓度紫杉醇或卡铂处理人卵巢癌细胞株CAOV3,发现随着药物作用时间的延长,存活的卵巢癌细胞survivin mRNA的表达量增加,而凋亡率的增加幅度减小,提示紫杉醇或铂类诱导的卵巢癌化疗中,卵巢癌细胞可能通过增加survivin表达而增强抗凋亡能力,从而产生耐药性。以上发现说明XIAP及survivin在卵巢癌细胞化疗耐药机制中是重要的决定因素。ZNF217与卵巢癌化疗耐药有无关系?对ZNF217进行基因调控后,卵巢癌的化疗敏感性会产生何种影响?其机制如何?这些问题都有待深入探讨。本研究的目的：(1)从蛋白水平上分析临床卵巢癌组织标本ZNF217的表达,同时采用ATP法检测卵巢癌组织对顺铂的化疗敏感性,以明确ZNF217的表达是否与顺铂化疗敏感性、卵巢癌分期、病理类型及分化程度有关；(2)比较不同卵巢癌细胞株ZNF217 mRNA和蛋白的表达水平,以了解顺铂耐药株和敏感株ZNF217的表达是否有明显差异；(3)通过RNA干扰和基因转染技术调控ZNF217基因的表达,以明确ZNF217表达水平对卵巢癌细胞顺铂化疗敏感性的影响,并从凋亡通路的角度探讨ZNF217影响化疗敏感性的机制。本研究旨在为阐明卵巢癌的化疗耐药机制提供理论依据,并为ZNF217作为卵巢癌潜在治疗靶点提供理论依据。材料和方法1、卵巢癌组织的来源：于2008年8月至2009年12月在深圳北大医院、深圳市人民医院和深圳市福田人民医院采集新鲜手术标本共60例,其中36例浆液性囊腺癌,9例粘液性囊腺癌,8例透明细胞癌,7例子宫内膜样癌,同时采用15例卵巢良性肿瘤及12例正常卵巢组织标本作对照；2、将卵巢癌组织制成癌细胞悬液,ATP法检测顺铂化疗敏感性；同时将卵巢组织制成石蜡切片,用于ZNF217蛋白免疫组织化学染色；3、培养卵巢癌细胞系A2780和A2780顺铂耐药株(A2780-DDP-R)、SKOV-3和SKOV-3顺铂耐药株(SKOV-3-DDP-R)、COC1和COC1顺铂耐药株(COC1-DDP-R), ATP法测定各耐药株的耐药指数(RI)；免疫荧光细胞化学明确ZNF217蛋白在卵巢癌细胞内的定位；RT-PCR技术和Western blotting技术分别检测ZNF217 mRNA和ZNF217蛋白的表达水平；4、构建pGenesil-ZNF217-siRNA重组质粒和pEGFP-N1-ZNF217过表达重组质粒,分别转染卵巢癌细胞系A2780-DDP-R和A2780,G418抗性筛选稳定转染的卵巢癌细胞系。5、培养卵巢癌细胞系A2780、pEGFP-N1-ZNF217/A2780、A2780-DDP-R、pGenesil-ZNF217-siRNA/A2780-DDP-R, ATP法测定顺铂对卵巢癌细胞的生长抑制率；流式细胞仪检测凋亡比率；Western blotting检测凋亡抑制蛋白XIAP和survivin的表达水平；分光光度法检测凋亡终末效应酶caspase3和caspase9的活性。6、统计学方法：全部数据均采用SPSS 13.0软件进行统计分析。非参数资料的比较采用非参数Kruskal-Wallis检验,相关分析采用等级资料的Spearman等级相关分析。计量资料用x±s表示。多组间计量资料比较采用多样本均数的One-Way ANOVA分析及基于One-Way ANOVA分析的LSD多重比较方法,两组间计量资料比较采用两独立样本均数的t检验。P<0.05为有统计学意义。结果1、卵巢癌组织对顺铂化疗的敏感性及ZNF217的表达耐药、部分敏感及高度敏感的卵巢癌组织均有ZNF217蛋白不同程度的表达,三者的差异有统计学意义(χ2=9.211,P=0.010), ZNF217蛋白表达与卵巢癌的顺铂化疗敏感性负相关(r=-0.394,P=0.002)；ZNF217蛋白在Ⅲ-Ⅳ期卵巢癌组织中的表达水平显著高于Ⅰ-Ⅱ期卵巢癌组织,差异有统计学意义(χ2=9.610,P=0.002),ZNF217蛋白表达与卵巢癌的临床分期有相关性(r=0.404,P=0.001)；在浆液性囊腺癌、粘液性囊腺癌、透明细胞癌和子宫内膜样癌这四种不同病理类型的卵巢癌组织中ZNF217蛋白表达水平无显著性差异(χ2=1.925,P=0.588),ZNF217蛋白表达与卵巢癌的病理类型无相关性(r=0.056,P=0.670)；不同分化程度的卵巢癌组织ZNF217蛋白的表达差异有统计学意义(χ2=9.934,P=0.007),ZNF217蛋白表达与卵巢癌的分化程度负相关(r=-0.410,P=0.001)；ZNF217蛋白在正常卵巢、卵巢良性肿瘤和卵巢癌组织中均有不同程度的表达,三者的差异有显著性(χ2=20.469,P=0.000),ZNF217蛋白表达与卵巢癌的发生密切相关(r=0.487,P=0.000)。2、卵巢癌顺铂化疗敏感株与耐药株中ZNF217的表达A2780对顺铂的IC50为8.1mg/L, A2780-DDP-R对顺铂的IC50为47.9mg/L, A2780-DDP-R对A2780的RI是5.9。SKOV-3对顺铂的IC50为9.6mg/L,SKOV-3-DDP-R对顺铂的IC50为40.1mg/L,SKOV-3-DDP-R对SKOV-3的RI是4.2。COC1对顺铂的IC50为4.8mg/L, COC1-DDP-R对顺铂的IC50为15.3mg/L, COC1-DDP-R对COC1的RI是3.2。该结果说明各卵巢癌耐药株对DDP具有明确耐药性。在免疫荧光实验中,发现各卵巢癌细胞株的细胞质及细胞核内均有ZNF217蛋白的表达,但在A2780、SKOV-3和COC1这三个敏感株中ZNF217蛋白主要分布于细胞质,而在三个耐药株A2780-DDP-R、SKOV-3-DDP-R以及COC1-DDP-R中ZNF217蛋白则主要分布于细胞核。RT-PCR和Western blotting技术分别检测卵巢癌细胞中ZNF217 mRNA和ZNF217蛋白的表达,结果显示三个耐药株表达水平较高,与敏感株的差异有统计学意义(F=20.303,P=0.000)(F=28.349,P=0.000)。3、调控卵巢癌细胞株中ZNF217基因的表达PCR技术扩增ZNF217基因全长序列,以pEGFP-N1质粒为载体,构建出pEGFP-N1-ZNF217过表达重组质粒。同时设计合成针对ZNF217的siRNA序列,以pGenesil-1质粒为载体,构建出pGenesil-ZNF217-siRNA重组质粒。将pEGFP-N1-ZNF217过表达重组质粒和pGenesil-ZNF217-siRNA重组质粒分别转染卵巢癌细胞株A2780和A2780-DDP-R。采用G418进行抗性筛选,获得分别稳定转染pEGFP-N1-ZNF217过表达重组质粒和pGenesil-ZNF217-siRNA重组质粒的卵巢癌细胞株pEGFP-N1-ZNF217/A2780和pGenesil-ZNF217-siRNA /A2780-DDP-R。通过RT-PCR和Western blotting技术鉴定细胞稳定转染成功。4、调控ZNF217基因表达对卵巢癌细胞株顺铂化疗敏感性的影响及机制顺铂对各组生长抑制率的差异有统计学意义(F=125.441,P=0.000)。顺铂对pEGFP-N1-ZNF217/A2780组的生长抑制率低于A2780组,提示过表达ZNF217可降低卵巢癌细胞对顺铂的化疗敏感性；顺铂对pGenesil-ZNF217-siRNA /A2780-DDP-R组的生长抑制率高于A2780-DDP-R组,提示通过RNA干扰降低ZNF217表达,可增强卵巢癌细胞对顺铂的化疗敏感性。以上结果说明ZNF217的表达水平是影响卵巢癌细胞顺铂耐药性的一个关键因子。流式细胞仪检测细胞凋亡率,顺铂诱导的各组细胞凋亡率的差异有统计学意义(F=78.688,P=0.000)。pEGFP-N1-ZNF217/A2780组的细胞凋亡率低于A2780组,提示过表达ZNF217可减少顺铂诱导的卵巢癌细胞凋亡；pGenesil-ZNF217-siRNA/A2780-DDP-R组的细胞凋亡率高于A2780-DDP-R组,提示通过RNA干扰降低ZNF217表达,可增强顺铂诱导的卵巢癌细胞凋亡。利用Western blotting技术检测凋亡抑制蛋白XIAP和survivin的表达水平变化,结果显示,在ZNF217高表达的卵巢癌细胞株中,XIAP和survivin的表达量明显上升,而在ZNF217低表达细胞中二者的表达量均较低(F=63.981,P=0.000)(F=79.008,P=0.000)。通过分光光度法检测caspase3和caspase9的活性,发现在ZNF217高表达的卵巢癌细胞中caspase3和caspase9的活性均降低,而在ZNF217低表达细胞中它们的活性均升高(F=16.664,P=0.001)(F=28.320,P=0.000)。以上结果说明,ZNF217诱导卵巢癌细胞耐药性是通过抑制细胞凋亡途径而发挥作用的,其中XIAP、survivin、caspase3和caspase9起了重要的作用。结论1、ZNF217蛋白的表达水平与卵巢癌组织对顺铂的化疗敏感性呈负相关；2、ZNF217蛋白在卵巢癌耐药株主要为核表达,而在卵巢癌敏感株则主要为浆表达；卵巢癌耐药株ZNF217为高表达,而卵巢癌敏感株则为低表达；3、ZNF217表达水平变化可以影响卵巢癌细胞的顺铂化疗敏感性,其表达水平越高,卵巢癌细胞对顺铂的化疗敏感性越低；4、ZNF217可以通过上调凋亡抑制蛋白XIAP和survivin的表达,进而下调凋亡终末效应酶caspase3和caspase9活性,最终抑制凋亡而导致卵巢癌细胞对顺铂的耐药性。更多还原

【Abstract】 BACKGROUNDOvarian cancer, a common gynecological tumor, has a high fatality in patients. Ovarian cancer is highly malignant, and easy to spread and transfer through blood and lymphatic system. The 5-year survival rate for advanced ovarian cancer patients is low, and it is mainly due to the chemotherapy drugs resistance. ZNF217 gene, a recently identified cancer gene, is located on chromosome 20ql3.2, the Kruppel-like transcription factors encoded by which is belong to the zinc finger protein family. ZNF217 gene is closely related to the occurrence and development of several tumors, such as ovarian cancer, mammary cancer, and lung cancer. We have previously found that the amplification degree of ZNF217 gene was correlated with clinical stages of ovarian cancer, the cancer cells’abilities of movement and invasion were apparently impaired after RNAi to ZNF217 gene, indicating that there is relationship between ZNF217 and the metastasis of ovarian cancer. Some investigator found that bZNF217 could decrease the apoptosis induced by adriacin, the adriacin sensitivity increase after inhibiting the ZNF217 expression by RNAi. But it is still not clear if there is some link between ZNF217 and chemotherapy drugs resistance.The chemotherapy drug resistance is an important reason for the failure of tumor therapy, and its main mechanism is the blocking-up of apoptosis pathway mediated by apoptotic genes. IAPs are a special anti-apoptosis family, including 8 members, XIAP, survivin, c-IAP1, livin, and so on. Most IAPs can block the apoptosis pathway through directly inhibiting the enzyme activity of caspase-3, caspase-7 and caspase-9, inducing the chemotherapy drug resistance. The over-expression of IAPs is related to the failure of tumor treatment. The expression of XIAP and survivin increased in ovarian cancer tissues, the over-expression of them can inhibit the apoptosis of ovarian cancer cells and is correlated with DDP and paclitaxel resistance. After the sense XIAP cDNA is transfected into DDP-sensitive ovarian cancer cells, the over-expression of XIAP can make Akt phosphorylate and then inactivate. The inactivation of Akt blocks the apoptosis induced by DDP. It is considered that the DDP sensitivity of ovarian cancer cells maybe related to the inhibition of caspase-3 activation by XIAP. Clinical data analysis shows that survivin is correlated with the paclitaxel resistance. The paclitaxel resistance increases 4-6 times after sense survivin cDNA is stably transfected into human ovarian cancer cell lines without survivin gene. It is found that the expression of survivin mRNA increase significantly, while the apoptosis rate does not increase correspondingly after stimulation by paclitaxel or carboplatin at low concentration, indicating ovarian cancer cells can enhance anti-apoptosis ability by increasing the expression of survivin, then improving the paclitaxel or carboplatin resistance. All above findings show both XIAP and survivin are important factors in the mechanism of chemotherapy drug resistance of ovarian cancer cells.Is there a close relationship between ZNF217 and clinical chemotherapy resistance? How does ZNF217 gene regulation affect on DDP sensitivity of ovarian cancer cells? And what is its mechanism? These questions have not been answered. The purposes of the study include:(1) determining if there is a close correlation between ZNF217 protein expression and chemotherapy sensitivity, clinical stages, pathological types and differentiation grades of clinical tissue samples of ovarian cancer by immunohistochemistry and detecting their DDP sensitivity by ATP method; (2) making clear if there is some difference in ZNF217 mRNA and ZNF217 protein expression between DDP-resistant and DDP- sensitive ovarian cancer cell lines; (3) identifying the effects of ZNF217 on the DDP sensitivity of ovarian cancer cells and its mechanism in the perspective of apoptosis pathway via RNAi and gene transfection. The study is aimed to clarify the mechanism of chemotherapy resistance in ovarian cancer, and to probably find a new therapeutic target point for ovarian cancer.MATERIALS AND METHODS1. The ovarian cancer tissues were taken from peking university Shenzhen hospital, Shenzhen people’s Hospital, and Shenzhen futian people’s hospital during 2008.8 to 2009.12. There were total 60 ovarian cancer samples, including 36 serous cystadenocarcinomas,9 mucous cystadenocarcinomas,8 clear-cell carcinomas and 7 endometrioid carcinomas. Two contrast groups respectively consisted of 15 benign ovarian tumors and 12 normal ovary tissues.2. The ovarian cancer tissues were made into cancer cell suspension, and the DDP sensitivity was determined with ATP method; the ovarian tissue paraffin sections were used to perform immunohistochemistry stain.3. Six ovarian cancer cell lines, including 3 DDP-sensitive lines (A2780, SKOV-3 and COC1) and 3 DDP-resistant lines (A2780-DDP-R, SKOV-3-DDP-R and COC1-DDP-R) were cultured. The resistance indexes (RI) of DDP-resistant cell lines were determined with ATP method. Intracellular localization of ZNF217 protein in the 6 ovarian tumor cell lines was detected by immunofluorescent cytochemistry. The expressions of ZNF217 mRNA and ZNF217 protein were determined by RT-PCR and Western blotting, respectively. 4. pGenesil-ZNF217-siRNA and pEGFP-N1-ZNF217 eukaryotic expression plasmids were constructed and transfected into A2780-DDP-R and A2780 cells, respectively, with cationic liposome. Under G418 selection, monoclones were selected and proliferated to establish stably-transfected cell lines.5. In 4 ovarian cancer cell lines, A2780, pEGFP-N1-ZNF217/A2780, A2780-DDP-R, pGenesil-ZNF217-siRNA/ A2780-DDP-R, growth inhibition rate were measured by ATP method, the apoptosis rate by flow cytometry, two apoptosis inhibitory proteins, XIAP and survivin by Western blotting, and the activity of two apoptosis effector enzymes, caspase3 and caspase9 by spectrophotography.6. All data were statistically analyzed with SPSS 13.0 software. Kruskal-Wallis test was used to compare nonparametric data, and Spearman correlation was used to analyze the correlation between rank data. Measurement data are shown as means±SE. One-way ANOVA test and LSD test were applied to compare data of more than two groups, and Independent samples t test to compare data of two groups. Concentration-inhibition curve was fitted by nonlinear regression model.RESULTS1. The DDP-sensitivity and the expression of ZNF217 protein in ovarian cancer tissueZNF217 protein was present in DDP-resistant, partially DDP-sensitive, and highly DDP-sensitive ovarian cancer tissues in different degrees, the difference among three groups was significant (x2=9.211, P=0.010), and a negative correlation was found between ZNF217 protein expression and the DDP-sensitivity (r=-0.394, P=0.002). The difference between I-II stages andⅢ-Ⅳstages was significant (x2=9.610, P=0.002), and there was a positive correlation between ZNF217 protein expression and clinical stages (r=0.404, P=0.001). ZNF217 protein expression was not significantly different in serous cystadenocarcinomas, mucous cystadenocarcinomas, clear-cell carcinomas and endometrioid carcinomas (x2=1.925, P=0.588), and there was no correlation between ZNF217 protein expression and pathological types (r=0.056, P=0.670). The difference of ZNF217 protein expression among normal ovarian tissues, benign ovarian tumours and ovarian cancer was significant (x2=20.469, P=0.000), and the ZNF217 protein expression was closely related to the occurrence of ovarian cancer (r=0.487, P=0.000)。ZNF217 protein was expressed in ovarian cancer tissues of different differentiation grades, the difference among three groups was significant (x2=9.934, P=0.007), and the ZNF217 protein expression was significantly related to the occurrence of ovarian cancer (r=-0.410, P=0.001)。2. ZNF217 expression in DDP-resistant and DDP-sensitive ovarian cancer cell linesThe IC50 values of DDP to A2780 and A2780-DDP-R were 18.1 mg/L and 47.9 mg/L (P< 0.01), respectively, that to SKOV-3 and SKOV-3-DDP-R were 9.6mg/L and 40.1 mg/L (P< 0.01), respectively, and that to COC1 and COC1-DDP-R were 4.8 mg/L and 15.3 mg/L (P< 0.01), respectively. The RIs of A2780-DDP-R, SKOV-3-DDP-R and COC1-DDP-R to DDP were 2.6,4.2 and 3.2, respectively. Laser scanning confocal microscopy showed that ZNF217 protein was mainly located in the cytoplasm of the DDP-sensitive cell lines, but it was in the nuclei of the DDP-resistant cell lines. RT-PCR and Western blotting showed that the expressions of both ZNF217 mRNA and ZNF217 protein were significantly higher in DDP-resistant cell lines than in DDP-sensitive cell lines.3. The ZNF217 gene regulation of ovarian cancer cell lines.Through amplifying the encoding sequence of ZNF217 gene and constructing it into the pEGFP-N1 plasmid, we successfully obtained the pEGFP-N1-ZNF217 eukaryotic expression plasmid. In addition, siRNA sequence targeting ZNF217 was also designed and cloned into the pGenesil-1 plasmid to construct the pGenesil-ZNF217-siRNA plasmid. The pEGFP-N1-ZNF217 and pGenesil-ZNF217 plasmids were transfected into A2780 and A2780-DDP-R cell lines, respectively. Then G418 was used to select stably-transfected cells. Stable transfection was certified via RT-PCR and Western blotting.4. The effects of ZNF217 gene regulation on the DDP-sensitivity of ovarian cancer cell lines and its mechanismThe growth inhibition rate was statistically different in four ovarian cancer cell lines (F=125.441, P=0.000). It was lower in pEGFP-N1-ZNF217/A2780 than in A2780, indicating ZNF217 over-expression can decrease DDP sensitivity. It was higher in pGenesil-ZNF217-siRNA/A2780-DDP-R than in A2780-DDP-R, indicating ZNF217 RNAi can increase DDP sensitivity. The results suggest ZNF217 expression level is a key factor to affect DDP sensitivity of ovarian cancer cells.The apoptosis rate of each cell lines was detected after DDP stimulation. The result showed that, the apoptosis rate was statistically different in four cell lines (F=78.688, P=0.000). It was lower in pEGFP-N1-ZNF217/A2780 than in A2780, indicating ZNF217 over-expression can decrease DDP-induced apoptosis. It was higher in pGenesil-ZNF217-siRNA/A2780-DDP-R than in A2780-DDP-R, indicating ZNF217 RNAi can increase DDP-induced apoptosis.Western blotting analysis was applied to detect the expression levels of XIAP and survivin, two apoptosis inhibitory proteins, and the result showed that, in ovarian cancer cell lines with higher levels of ZNF217, the expression level of XIAP and survivin increased significantly (F=63.981, P=0.000) (F=79.008, P=0.000).The activities of caspase3 and caspase9 were measured by spectrophotography. It was found that, in ovarian cancer cell lines with higher levels of ZNF217, the activities of both caspase 3 and caspase 9 decreased significantly, while in ovarian cancer cell lines with lower levels of ZNF217, their activities increased significantl (F=16.664, P=0.001) (F=28.320, P=0.000). These result suggested that, ZNF217 induced DDP-resistance in ovarian cancer cells through inhibiting apoptosis pathway, and XIAP, survivin, caspase3 and caspase9 were the key elements regulated by ZNF217 in the apoptosis pathway.CONCLUSIONS1. There is a negative correlation between ZNF217 protein expression level and the DDP sensitivity of ovarian cancer tissues.2. ZNF217 protein is mainly located in the cytoplasm of the DDP-sensitive cell lines, while in the nuclei of the DDP-resistant cell lines. The expressions of both ZNF217 mRNA and protein were significantly higher in DDP-resistant cell lines than in DDP-sensitive cell lines.3. The ZNF217 expression level can influence the DDP resistance of ovarian cancer cells. The higher is the ZNF217 expression level, the lower is the DDP sensitivity.4. ZNF217 induces the DDP resistance in ovarian cancer cells through up-regulating the expression of XIAP and survivin and down-regulating the activities of caspase3 and caspase9.更多还原