乳腺癌相关基因异常甲基化的临床实验研究

【作者】 符德元；

【导师】 邵志敏； 陆劲松； 柳光宇；

【作者基本信息】 复旦大学 ， 肿瘤学， 2009， 博士

【摘要】 第一部分:Sox17基因在乳腺癌中的表达、异常甲基化及其与Wnt/β-Catenin通路关系的研究目的:Sox17是一种转录因子,具有广泛的生物学功能,可以在经典Wnt/β-catenin信号传导通路中担当拮抗因子的作用。本研究的目的是在乳腺癌细胞株及组织标本中探讨Sox17基因表达、甲基化状态及其潜在的临床意义。初步研究Sox17与β-catenin基因表达之间的关系,探讨Sox17基因在Wnt/β-catenin信号通路中的作用。方法:运用RT-PCR和实时定量RT-PCR检测乳腺癌细胞株在去甲基化药物5-aza-dC处理前后的Sox17、β-catenin基因mRNA的表达水平,Western blot检测其蛋白表达情况。运用小干扰RNA（small interfering RNA,siRNA）技术对两株正常表达Sox17mRNA的乳腺癌细胞株SK-BR-3和Bacp-37进行Sox17和β-catenin RNA干扰。运用亚硫酸氢盐测序（BSP）和甲基化特异性PCR（MSP）法对Sox17基因甲基化状态进行分析。此外,在31例配对的乳腺癌组织和癌旁标本中,对Sox17、β-catenin基因mRNA的表达水平进行定量检测,并对Sox17基因mRNA的表达水平与其甲基化状态之间的相互关系进行分析。最后,在113例乳配对腺癌组织和的癌旁标本中,运用MSP法对Sox17基因甲基化状态进行了检测,分析Sox17基因甲基化与乳腺癌主要临床病理特征参数之间的关系。结果:在5株不同的乳腺癌细胞株中和23/31乳腺癌组织标本中,Sox17mRNA的表达水平显著减少,且与Sox17基因在这些细胞和组织标本中的甲基化状态显著相关。在经去甲基化药物5-aza-dC处理后,5株细胞株的Sox17 mRNA的表达水平显著升高;去甲基化药物在使Sox17表达恢复的同时,使β-cateninmRNA表达水平呈下降趋势。此外,在正常表达Sox17的SK-BR-3和Bacp-37细胞株中,siRNA介导的Sox17表达降低可提高β-catenin的表达水平。在31例配对组织样本中,Sox17和β-catenin mRNA表达水平也存在着显著性差异（P＜0.001）。Sox17甲基化可在74.3%（84/113）的乳腺癌组织和31.9%（36/113）的配对癌旁组织中检测到,两者存在显著差异（P＜0.0001）。分析Sox17甲基化与乳腺癌主要临床病理特征显示,Sox17甲基化与乳腺癌临床肿瘤分期（P=0.028）及淋巴结转移（P=0.013）密切相关。结论:结果表明,在乳腺癌中,启动子甲基化是导致Sox17基因表达沉默的一个重要机制,而且Sox17基因的功能失活可能有助于Wnt信号通路的异常激活。在乳腺癌发生和发展过程中,Sox17甲基化可能是一个有价值的分子标志。第二部分:EphA5基因在乳腺癌中的表达及异常甲基化的研究目的:为了鉴别在乳腺癌中因基因甲基化状态改变而发生功能变化的新基因,根据我们先前的甲基化芯片结果分析表明,EphA5基因在乳腺癌组织标本中呈现高甲基化状态。本研究的目的是在乳腺癌细胞株、乳腺癌组织及其配对的癌旁组织中进一步探讨EphA5基因的表达情况、甲基化状态以及潜在的临床意义。方法:运用RT-PCR和实时定量RT-PCR检测乳腺癌细胞株在去甲基化物5-aza-dC处理前后的EphA5基因mRNA表达水平变化,运用western blot检测其蛋白表达变化情况。运用亚硫酸氢盐测序（BSP）和甲基化特异性PCR（MSP）对EphA5基因甲基化状态进行分析。此外,在31例乳腺癌组织和配对的癌旁标本中,对EphA5基因mRNA的表达水平进行定量检测,并对其表达水平和甲基化状态之间的相互关系进行分析。最后,在117例乳腺癌组织和配对的癌旁标本中,运用MSP法对EphAS基因甲基化状态进行了检测,分析EphA5基因甲基化与乳腺癌主要临床病理特征参数之间的关系。结果:EphA5基因在5株乳腺癌细胞株表达缺失。在去甲基化药物5-aza-dC处理后,EphA5基因的mRNA和蛋白表达水平显著上升。BSP和MSP检测表明,EphA5基因表达减少与其甲基化显著相关。在31例乳腺癌组织及其配对的癌旁标本中,21/31（67.7%）癌旁组织EphA5mRNA表达高于其在癌组织中表达2倍以上（P＜0.001）。并且Epha5甲基化与其mRNA表达变化显著相关（P=0.017）。在117例配对组织标本中,EphA5基因甲基化可在64.1%（75/117）的乳腺癌组织和28.2%（33/117）的配对癌旁组织检测到（P＜0.001）,EphA5甲基化状态与乳腺癌组织学分级、淋巴结转移与否、孕激素受体阳性与否显著相关（P=0.024,0.004,0.008）.结论:我们的研究结果表明,在乳腺癌中EphA5基因是一个潜在的,因启动子区CpG岛甲基化而发生表达沉默的基因。在乳腺癌发生和发展过程中,EphA5甲基化可能是一个有价值的分子标志。第三部分:多基因异常甲基化与乳腺癌主要临床病理特征关系的初步研究目的:基因启动子区异常甲基化导致的分子生物学特性改变是乳腺癌的一个普遍现象。但是,其对乳腺癌进展的影响及潜在的预测作用仍未完全明了。我们此研究的目的是分析乳腺癌中8个重要的、具有不同致癌机制的相关基因甲基化情况,评价这些基因甲基化与乳腺癌主要临床病理特征之间的关系,并在此基础上进一步探讨在血浆循环DNA中检测这些基因甲基化对乳腺癌潜在的诊断价值。方法:在106例配对的乳腺癌及癌旁组织及另96例血浆标本中,运用多重巢式甲基化特异性PCR法,对RASFF1A,APC,MGMT,GSTP1,p16,ESR1,RAR-β₂,BRCA1这8个抑癌基因的甲基化状况进行检测、分析。结果:这8个基因的甲基化频率从MGMT基因的18.9%到RASSF1A基因的77.4%。在乳腺癌患者组织标本中,至少有一个基因发生甲基化的为94.3%;每个标本的平均甲基化数在乳腺癌及其配对的癌旁组织中分别为3.53和1.32,具有显著差异;与乳腺癌临床病理特征关系最为密切的为组织学分级,APC,MGMT,p16和RAR-β₂四个基因甲基化均与其显著相关（P=0.014,0.025,0.002和0.026）;乳腺癌中同时存在的多个基因甲基化与肿瘤较大、具有较高的组织学分级、伴有淋巴结转移和雌激素受体阴性等显著相关。在血浆标本中,86.2%（50/58）的肿瘤患者至少存在一个基因甲基化;同没有基因甲基化的患者相比,有1～3个基因甲基化的患者诊断为乳腺癌的风险为4.3倍（95%CI 1.4～13.4,P=0.01）:当超过3基因甲基化时,风险升至20.1（95%CI 5.9-69.2,P＜0.0001）。结论:本组8个基因的甲基化情况与乳腺癌重要的临床病理特征显著相关,同时存在多个基因甲基化预示着乳腺癌具有侵袭性表型。在血浆循环DNA中联合检测这些表观遗传学标志对乳腺癌风险预测具有重要价值。更多还原

【Abstract】 Part 1:The Canonical Wnt Antagonist Sox17 Is Epigenetically Inactivated by Promoter Methylation in Human Breast CancerObjective:Sox17 is a transcription factor involved in many developmental processes and can act as an antagonist of canonical Wnt/β-catenin signaling pathway. This study was aimed to investigate the relationship between Sox17 gene expression and its methylation status,and its potential role in Wnt/β-catenin signaling in breast cancer.Methods:The expression levels of Sox17 andβ-catenin were examined by RT-PCR and real-time PCR in seven breast cancer cell lines treated with or without demethylation agent 5-aza-dC and 31 paired breast tissue samples.Methylation status was detected by bisulfite sequencing and methylation-specific PCR.Correlation of Sox17 methylation status with clinicopathologic characteristics was evaluated in 113 breast cancers.Results:The expression level of Sox17 mRNA was dramatically decreased in five different breast cancer cell lines and 23 of 31 primary breast tumor samples, which significantly correlated with its methylation status.Aider treated with 5-aza-2’-deoxycytidine（5-aza-dC,a demethylation agent）,the expression levels of Sox17 mRNA and protein were obviously increased.Restored expression of Sox17 by 5-aza-dC treatment decreased the expression level ofβ-catenin in breast cancer cell lines.Furthermore,small interfering RNA（siRNA）-mediated knockdown of Sox17 in SK-BR-3 and Bacp-37 cells enhancedβ-catenin expression.In 31 paired tissue samples,a significant difference between the expression level of Sox17 andβ-catenin was also observed（P＜0.001）.Clinically,Sox17 methylation was detected in 74.3% breast tumors（84/113） and 31.9%（36/113） paired normal tissues respectively （P＜0.0001）.Sox17 methylation was also associated with tumor stage（P=0.028） and lymph node metastasis（P=0.013）.Conclusion:These findings indicate that silencing of Sox17 due to promoter hypermethylation is a frequent event and may contribute to aberrant activation of Wnt signaling in breast cancer.Sox17 may be a valuable biomarker for the study of breast cancer carcinogenesis and progression. Part 2:Frequent Epigenetic Inactivation of Receptor Tyrosine Kinase EphA5 by Promoter Methylation in Human Breast CancerObjective:In an effort to identify new genes that are functionally affected by altered methylation status in breast cancer,a microarray based methylation analysis showed that EphA5 gene is higher hypermethylated in tumor samples.The aim of the present study was to investigate EphA5 gene expression profiles,methylation status, and clinical significances in breast cancer.Methods:Gene expression level of EphA5 was detected by RT-PCR and realtime RT-PCR with or without 5-aza-2’-deoxycytidine treated in breast cancer cell lines. Methylation status was tested by Bisulfite genomic sequencing and MSP.Further, correlation between expression level and methylation status of EphA5 gene were assessed in 31 paired tissue samples.Finally,the methylation status of EphA5 gene was examined in total of 117 paired tissue samples by MSP assay and the correlation with patients’ clinicopathologic features were also evaluated.Results:The expression level of EphA5 mRNA was dramatically decreased in five different breast cancer cell lines.Aider treated with 5-aza-2’-deoxycytidine （5-aza-dC,a demethylation agent）,the expression levels of EphA5 mRNA and protein were obviously increased.Bisulfate sequencing and methylation-specific PCR detection showed that decreased expression of EphA5 was associated with its methylation status.We also found a significant correlation（P=0.017） between reduction of EphA5 mRNA abundance and aberrant methylation of EphA5 in 31 paired tissue samples.In clinical samples,EphA5 methylation was detected in 64.1% breast tumors（75/117） and 28.2%（33/117） paired normal tissues respectively （P＜0.001）,which was associated with higher tumor grade（P=0.024）,lymph node metastasis（P=0.004）,and PR negative status（P=0.008）.Conclusion:Our findings indicate that EphA5 is likely a potential target for epigenetic silencing in primary breast cancer.EphA5 may be a valuable molecular marker of breast cancer carcinogenesis and progression. Part 3:Multiple Gene Methylation in Chinese women with Sporadic Breast CancerObjective:Multiple gene methylation is common in breast cancer and may be a useful biomarker for breast cancer detecting.However,the correlation between multiple genes methylation status and clinicopathological parameters,and diagnostic value in Chinese breast cancer women are unknown.Material and Methods:Using mutiplex nested methylation specific PCR,we analyzed methylation status of eight critical genes:RASFF1A,APC,MGMT,GSTP1, p16,ESR1,RAR-β₂ and BRCA1,in 106 paired breast tumors and corresponding normal tissues,and 96 plasma DNA samples including 38 benign breast diseases and 58 breast cancer patients.Results:The frequency of methylation of these genes varied from 18.9%for MGMT to 77.4%for RASSF1A.Methylation in at least one of the genes was found in 94.3%of the breast cancer.And the mean number of genes hypermethylated in each tumor and paired normal breast tissues was 3.53 and 1.32 respectively.The strongest and most consistent association observed of specific gene methylation was with higher tumor grade,there are four genes APC,MGMT,RAR-β2 and p16 reached statistical significance.High frequency simultaneous methylation of multiple genes was more often in these with higher tumor grade,lymph node metastasis,or ER negative patients.In plasma samples,86.2%（50/58） tumors DNA showed hypermethylation of at least one of the eight genes.Logistic regression models analysis showed that the odds ratio for diagnosis of breast cancer was 4.3（95%CI 1.4～13.4,P=0.01） in patients with 1～3 methylated gene and 20.1 in patients with more than 3 genes methylation（95%CI was 5.9-69.2,P＜0.0001）.Conclusions:Hypermethylation of these genes may be linked to various known clinicopathological features of breast cancer in Chinese women,and increasing multiple gene methylation in tumors may indicate a aggressive phenotype for breast cancer.Detection of hypermethylation in plasma DNA using a combination of these epigenetic markers may be useful for identifying patients at high risk for breast cancer.更多还原