【作者】 唐兆前；

【导师】 唐步坚； 李力；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2010， 博士

【摘要】 卵巢上皮癌在妇科恶性肿瘤中致死率最高。手术后,以铂类为基础的联合化疗是其主要的治疗方法。但随着化疗的进行,患者最终获得难以治愈的多药耐药,因而其五年生存率一直徘徊在30-50%。为了更好地认识肿瘤抑制基因在卵巢癌多药耐药中的机制,我们对卵巢癌卡铂耐药基因差异表达谱芯片进行了肿瘤抑制基因的筛选及验证,对相关的肿瘤抑制基因启动子区进行了甲基化检测以及突变基因筛选研究。并在组织标本中进行了临床意义的探讨。第二章卵巢癌卡铂耐药细胞与非耐药细胞差异表达基因的验证目的:筛选卵巢癌卡铂耐药表达谱基因芯片中卡铂耐药细胞系(SKOV3-CB)较其亲本(SKOV3)细胞系差异表达下调的肿瘤抑制基因,并验证其差异表达。方法:应用分子生物信息学方法,分析卵巢癌卡铂耐药表达谱基因芯片中卵巢癌SKOV3-CB细胞系较SKOV3细胞系差异表达下调两倍以上的基因1554条,筛选肿瘤抑制基因。应用real-time PCR技术对基因芯片中差异表达基因检测验证。结果:从卵巢癌卡铂耐药表达谱基因芯片中筛选出RNASET2,VHL,DLG1,COPS2,NOL7,GGNBP2, RBL1, S100A2, PARG1,PERP,TCF3,DNAJA3,PCGF2,ING1,CYLD,RARRES3,RBBP8等17个肿瘤抑制基因,荧光定量PCR技术差异表达验证结果示:RNASET2,VHL, COPS2,NOL7, RBL1, S100A2, PARG1,PERP,TCF3,DNAJA3, ING1,CYLD,RARRES3,RBBP8等基因差异表达下调,下调倍数分别为24.47、65.24、10.81、21.30、72.94、5.95、27.13、1.57、1.32、12.93、46.8、7.57、14.9、14. 00、23.98。GGNBP2,PCGF2,DLG1等基因差异表达上调,上调倍数分别为5.76,2.19,17.1,基因芯片差异表达结果与real-time PCR所验证表达下降趋势的结果一致率为82.35%,下调两倍以上的基因一致率为64.70%。结论:利用基因芯片技术分析基因差异表达结合real-time PCR对其检测结果验证是一种有效的方法,多肿瘤抑制基因参与卵巢癌卡铂耐药的机制。第三章卵巢癌卡铂耐药细胞与非耐药细胞差异表达基因甲基化检测目的:探讨卵巢癌卡铂耐药相关肿瘤抑制基因启动子区甲基化状态与卵巢癌卡铂耐药表达下调的关系。方法:应用分子生物信息学方法,分析肿瘤抑制基因启动子区CPG岛并设计出引物,采用甲基化特异性PCR(MS-PCR)法或亚硫酸盐测序法(bisulphite sequencing)检测SKOV3-CB、SKOV3中的耐药相关肿瘤抑制基因启动子区甲基化状态。结果:表达下调的14个基因(VHL, COPS2, NOL7,RANSET2, RBL1, S100A2, PARG1, PERP, TCF3, DNAJA3, ING1, RARRES3, RBBP8,CYLD)中,有10个基因(VHL, COPS2, NOL7, RBL1, PARG1, PERP, DNAJA3, ING1, RNASET2,CYLD)启动子区能够设计出引物,其中CYLD未检出结果,后改用亚硫酸盐测序法检测。MS-PCR检测结果显示,SKOV3-CB、SKOV3的ING1、DNAJA3、VHL甲基化引物均能够扩增出产物,未甲基化引物均不能扩增出产物;COPS2、PERP甲基化引物均不能够扩增出产物,未甲基化引物均能够扩增出产物;RNASET2,PARG1、NOL7、RBL1甲基化引物与未甲基化引物均能够扩增出产物,CYLD重亚硫酸盐测序提示在SKOV3-CB及SKOV3细胞系均为未甲基化状态。结论:卵巢癌卡铂耐药相关肿瘤抑制基因启动子区无超甲基化,甲基化机制不是引起卵巢癌卡铂耐药相关肿瘤抑制基因差异表达下调的原因。第四章卵巢癌卡铂耐药细胞与非耐药细胞差异表达基因突变检测目的:筛查可能的基因突变,探讨其与卵巢卡铂耐药以及相关肿瘤抑制基因表达下调的关系。方法:以亲本细胞系(SKOV3)为对照,在卵巢癌卡铂耐药细胞系(SKOV3-CB),应用单链构象多态性分析(SSCP)检测RNASET2,VHL, COPS2, NOL7, PARG1, RBL1, PERP, DNAJA3, ING1, CYLD等10个基因RT-PCR扩增片段的突变。结果:以SKOV3细胞系为对照,在SKOV3-CB细胞系中,SSCP检测发现DNAJA3,PERP基因聚丙烯酰胺电泳带型存在差异;RNASET2,VHL,COPS2, NOL7, PARG1, RBL1,ING1, CYLD等基因未检测到差异带型。经测序发现PERP第三外显子1093,1160,1222位点上分别存在A→G突变;1083位点及1085,1086位点,分别存在T,C,C,碱基缺失。DNAJA3第八个外显子87位点处存在A→T突变。结论:DNAJA3,PERP的基因突变以及PERP基因的碱基缺失与卵巢癌卡铂耐药相关,可能是导致其表达下调的原因,宜进一步深入研究。第五章卵巢癌卡铂耐药相关肿瘤抑制基因表达与临床病理的关系的研究目的:探讨卵巢癌卡铂耐药相关肿瘤抑制基因表达的临床意义方法:Trizol一步法提取卵巢肿瘤患者卵巢组织总RNA,应用RT-PCR检测85例卵巢组织的RNASET2、VHL、PARG1、CYLD、COPS2、PERP、DNAJA3、ING1、NOL7、RBL1基因mRNA表达,其中卵巢癌49例,卵巢良性组织21例,卵巢正常组织15例。结果: RT-PCR结果显示RNASET2、ING1、PARG 1、RBL1、NOL7基因在正常卵巢组织、卵巢良性肿瘤组织、卵巢癌组织中均有一定程度表达;VHL、PERP、COPS2、CYLD基因在正常卵巢组织、良性肿瘤中、卵巢癌组织中均有一定程度表达,但在部分卵巢癌组织中表达沉默,分别为4(4/49)、3(3/49)、2(2/49)、32(32/49)例;采用Fisher确切概率法统计分析,提示RNASET2、ING1、PARG 1、RBL1、NOL7、VHL、PERP、COPS2基因在卵巢癌中的表达沉默阳性率与在卵巢正常组织及卵巢良性肿瘤中的表达沉默阳性率比较差异无意义。CYLD、DNAJA3基因在卵巢癌中的表达沉默阳性率与在卵巢良性肿瘤组织与正常卵巢组织中的表达沉默阳性率比较差异有显著意义(P分别为0.00、0.00;0.00,0.00)。VHL等10个基因表达与卵巢癌临床病理的关系Fisher确切概率法统计分析,提示CYLD在FIGO I-II与III-IV期存在差异有意义外(P<0.05),其余基因差异均无意义(P均>0.05)。Log Rank分析发现卵巢癌组织VHL、PERP、CYLD基因表达阳性患者与阴性患者的生存时间比较,差异均无统计学意义(P分别为0.183、0.493、0.271)。COPS2基因表达阳性患者与阴性患者的生存时间比较,差异有统计学意义(P=0.005)。COX回归模型多因素生存分析提示FIGO分期、病理分级进入模型(P分别为0.045、0.028);淋巴结转移及COPS2、PERP、CYLD、ING1基因表达沉默等参数均未进入模型(P分别为0.907、0.476、0.754、0.728、0.304)。结论:结论CYLD、DNAJA3基因表达沉默可作为卵巢癌与卵巢良性肿瘤及正常卵巢组织鉴别诊断的一个潜在的指标,COPS2、PERP、CYLD、ING1不能作为卵巢癌独立的预后因素。更多还原

【Abstract】 Mortality of epithelial ovarian cancinoma is highest in gynecological malignant tumor. After the surgery, chemotherapy is the major treatment methods based on the combination of platinum chemotherapy. But ultimately, it is difficult to cure patients because of resistance to ovarian carcinoma.So its 5 years survival rate is always 30-50%. In order to better understand the mechanism of tumor suppressor genes in multi-drug resistance of ovarian cancinoma ,tumor suppressor genes related to carboplatin resistance were selected and verified from carboplatin resistance microarray. Promoter region methylation were detected and gene mutation were screened for tumor suppressor genes related to carbo-resistance.Clinical implication of tumor suppressor genes were futher explored in ovarian carcinoma tissue . Part two Verification of differential expression genes between carbo-resistance cell line and its parental cell line of ovarian carcinomaObjective: To select tumor suppressor gene from carboplatin resistance microarray , which its downregulation expression were verified between SKOV3 carboplatin resistance cell line and its parental SKOV3 cell line .Methods: tumor suppressor genes were selected from carboplatin resistance microarray ,which 1554 genes were analysized by bioinformation methods. differential expression of tumor suppressor gene which were two more folds in microarray between resistance SKOV3 cell line and SKOV3 cell lines were verified respectively using real-time RT-PCR。Results: . 17 tumor suppressor genes which were RNASET2,VHL,DLG1,COPS2,NOL7,GGNBP2, RBL1, S100A2, PARG1,PERP,TCF3,DNAJA3,PCGF2,ING1,CYLD,RARRES3,RBBP8 were selected from the microarray.Of those,differential expression of genes such as RNASET2,VHL, COPS2,NOL7, RBL1, S100A2, PARG1,PERP,TCF3,DNAJA3, ING1,CYLD,RARRES3,RBBP8 were downregulation whose folds were respectively 24.47、65.24、10.81、21.30、72.94、5.95、27.13、1.57、1.32、12.93、46.8、7.57、14.9、14. 00、23.98 .but differential expression of the three genes such as GGNBP2,PCGF2,DLG1 were upregulaion whose folds were 8.0,7.23,1.24 respectively. Accordance rate of the differential experssion downregulation result which were one more folds and two more folds between experimental verification to microarray and microarray respectively were 82.35%,64.70%。Conclusion:It is a effective method to analyse differential expression of gene which the results were verificated to microarray by real-time PCR technology and multiple tumor suppressor gene involved in the mechanism of resistance of ovarian carcinoma. Part three Methylation detection of differential expression gene between carbo- resistance cell line and parental cell line of ovarian carcinomaObjective: To observe the tumor suppressor gene promoter methylation status related to carboplatin resistance of ovarian carcinoma and explore its relationship with expression downregulation in carboplatin resistance of ovarian carcinoma.Methods: Using molecular bioinformatics methods, promoter CPG island of tumor suppressor gene were analysized to design primers for MS-PCR or bisulphite sequencing .And promoter methylation status of tumor suppressor genes associated with carboplatin resistance were detected between SKOV3-CB and SKOV3 cell line of ovarian carcinoma using methylation-specific PCR (MS-PCR) or bisulphite sequencing method.Results: 14 genes (VHL, COPS2, NOL7, GGNBP2, RBL1, S100A2, PARG1, PERP, TCF3, DNAJA3, ING1, RARRES3, RBBP8,CYLD) whose differential expressions were downregulation in SKOV3-CB cell line comparing with SKOV3 cell line. Of those ,10 genes (VHL, COPS2, NOL7, RBL1, PARG1, PERP, DNAJA3, ING1, RNASET2,CYLD) could be designed primers for MS-PCR in its promoter region. MS-PCR showed that PCR products of could be amplified by the ING1, DNAJA3, VHL methylation primers and PCR products could not be amplified by the unmethylated primers in both SKOV3-CB andSKOV3 cell line; PCR products could not be amplified by COPS2, PERP methylation primers and PCR products could be amplified by the unmethylated primers in both SKOV3-CB andSKOV3 cell line; PCR products could be amplified by both RNASET2,PARG1、NOL7、RBL1 methylation primers and unmethylated primers in the two cell lines,CYLD which could not be amplified by MS-PCR primers were amplified by bisulphite sequencing and its promoter region were detected to be unmethylated in the two cell lines.Conclusion: No hypermethylation were detected in promoter region of the tumor suppressor genes related to carboplatin-resistance of ovarian carcinoma ,which differential expression downregulation of the tumor suppressor genes is not caused by methylation mechanism . Part four Mutation detection of differential expression gene between carboplation resistance and parental cell line of ovarian carcinomaObjective: Screening of possible genetic mutations, to explore relationship with carboplatin resistance of ovarian and expression downregulation of related tumor suppressor genesMethods: The parental cell line (SKOV3) as control, in the carboplatin-resistant ovarian cancer cell line (SKOV3-CB), single-strand conformation polymorphism analysis (SSCP) were applied to detect RT-PCR amplified fragments of RNASET2, VHL, COPS2, NOL7, PARG1, RBL1 , PERP, DNAJA3, ING1, CYLD10 genes so as to find genetic mutation.Results: In the SKOV3-CB cell lines, differential types in polyacrylamide gel electrophoresis of DNAJA3, PERP gene were detected by SSCP in contrast to SKOV3 cell lines, NO differential types of RNASET2, VHL, COPS2, NOL7, PARG1, RBL1, ING1, CYLD genes were detected in polyacrylamide gel electrophoresis between SKOV3-CB and SKOV3 . Respectively there were A→G pot mutation in 1093,1160,1222 locous and T,C,C loss in 1083,1085,1086 locous of the third exon of PERP.There was A→T pot mutation in 87 locous of the sequence of the eighth exon of DNAJA3.Conclusion: DNAJA3, PERP gene mutation may be related to carboplatin resistance of ovarian carcinoma and to expression downregulation of related tumor suppressor genes . Should be further investigated Part five The clinical research of expression of related carbo-resistance tumor suppressor genes of ovarian carcinomaObjective:To investigate the clinical implications of expression of ovarian carcinoma carboplatin resistance-related tumor suppressor gene.Methods: The total RNA of the ovarian tissues was extracted by one-step Trizol extraction method. Expressions of genes which were RNASET2、VHL、PARG1、CYLD、COPS2、PERP、DNAJA3、ING1、NOL7、RBL1 were detected in ovarian tissues of 85 patients using RT-PCR,which included 49 patients with ovarian carcinoma, 21 patients with benign ovarian tumors,15 patients with normal ovary.Results:RT-PCR results show that there were expressions of RNASET2、ING1、PARG 1、RBL1、NOL7 gene in ovarian carcinoma tissues ,ovarian benign tumor tissues and normal ovarian tissues and there were expression of VHL、PERP、COPS2、CYLD gene in normal ovarian tissues ,ovarian carcinoma tissues and ovarian benign tumor tissues except expression silence in some ovarian carcinoma tissues which there were 4(4/49)、3(3/49)、2(2/49)、32(32/49)cases respectively.There are no difference for positive ratio of expression silence of RNASET2、ING1、PARG 1、RBL1、NOL7、VHL、PERP、COPS2 gene in between ovarian carcinoma tissues and NOT ovarian carcinoma tissues(P were 1.00、1.00、1.00、1.00、1.00、0.11、0.19、0.33 respectively)and there were difference for Positive ratio of expression silence of CYLD、DNAJA3 gene in between ovarian carcinoma tissues and NOT ovarian carcinoma tissues(P were 0.00、0.00 respectively) with chi square test. There were no difference for survival time between positive expression and negative experrsion of VHL,PERP,CYLD gene (P were 0.183、0.493、0.271 respectively) and there was difference for survival time between positive expression and negative experrsion of COPS2 gene in ovarian carcinoma patients with Log Rank(P=0.005). COX regression show that FIGO stage、differentiation involved in the model(P were 0.045、0.028 respectively)and Lymph node metastasis and expression silence of COPS2、PERP、CYLD、ING1 gene did not involved in the model(P were 0.907、0.476、0.754、0.728、0.304 respectively).Conclusion:Expression silence of CYLD、DNAJA3 gene maybe were potential marker for differential diagnosis in ovarian tumors and expression silence of COPS2、PERP、CYLD、 ING1 gene were not independent prognostic fators for ovarian carcinoma.更多还原