乳腺癌全基因组甲基化差异分析与重要功能基因的临床验证
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摘要
总体研究目的和意义：根据国际抗癌协会的资料统计,乳腺癌是女性发病率较高的恶性肿瘤之一,约占23%,全世界每年约120万妇女患病,50万人死亡,发病率和死亡率居妇女各类恶性肿瘤之首,并以每年0.3%-8%的速度增长。在中国妇女乳腺癌的发病率呈上升趋势,成为危害女性健康的主要杀手,占女性恶性肿瘤的32%。据全国肿瘤登记中心收到的共计49个肿瘤登记地区上报的2006年恶性肿瘤登记资料显示,乳腺癌作为中国女性发病率第一位的恶性肿瘤,其发病率为23.3/10万,死亡率也已上升到4.71/10万。目前国际认可的最常用的乳腺癌风险预测方法是Gail模型,然而其对散在乳腺癌预测精确度只有大约58-59%,而且对亚洲人的适用性仍未知。乳腺癌易感基因BRCA1和BRCA2突变与家族性乳腺癌密切相关,但只占5-10%的乳腺癌。近期的全基因组关联研究(GWASs)已经确定了基因多态性与乳腺癌风险相关,一组10个SNPs有59.7%的预测精确度。因此,已知乳腺癌的环境和遗传危险因素在预测一个妇女患病风险上是有限的。早期诊断和早期治疗是提高乳腺癌患者生存率与生活质量的关键；然而目前用于乳腺癌早期诊断的手段主要是自我检查和影像学检查,这些手段有约30%的漏诊率。而且即便是Ⅰ、Ⅱ期乳腺癌仍有15%-25%最终会发生远处转移。因此,进一步明确乳腺癌发生发展的分子机制、寻找新的治疗靶点以及探索科学的综合治疗模式以提高早诊早治率,是乳腺癌研究的重要内容。多年来人们一直在探索通过外周血检测肿瘤的最佳标志物,并试图通过这一便捷、易被医患双方所接收的方式达到肿瘤预警、早期诊断、监控病情、判断预后的目的,然而迄今尚无理想的结果。
恶性肿瘤的发生是环境与遗传因素作用下多阶段、多步骤、多基因异常累积的结果,其中最主要的环节是癌基因的激活和抑癌基因的失活。根据经典肿瘤发生的“二次打击理论”,认为抑癌基因的失活有两条途径,即基因内突变和染色质丢失。然而随着研究的深入,人们发现某些恶性肿瘤DNA序列完整,并未有突变、缺失,“二次打击理论”无法解释抑癌基因何以失活。这种用传统遗传学无法解析的现象,催生了与遗传学相对应的另一门新兴学科-一表观遗传学。DNA甲基化是一种重要的遗传外修饰,是表观遗传学的重要组成部分,是调节基因组功能的重要手段。DNA甲基化主要发生在G/C含量丰富的CpG二核苷酸位点(CpG岛),CpG岛对基因的表达起着调控作用,具有特殊的意义。在人类和多数哺乳动物,DNA甲基化仅影响DNA链上鸟嘌呤前的胞嘧啶。正常细胞DNA序列CpG位点约有70-80%发生了甲基化,而大多数CpG岛内CpG位点完全无甲基化。DNA甲基化在基因表达的调控中具有重要作用。近期研究直接分析了基因启动子的CpG岛甲基化,并与LOH或突变分析相结合,证实基因启动子高甲基化是抑癌基因失活的第三种机制,并且在某些情况下是抑癌基因失活的唯一机制,并参与多种肿瘤的发生、发展过程。如Yan等利用外显子测序和甲基化芯片分析发现在急性单核细胞白血病中DNMT3A高突变率,且具有该突变的同时伴随有该基因DNA异常甲基化的出现,进一步验证了启动子甲基化在肿瘤研究中的重要地位。
因此,大量关于启动子异常甲基化与乳腺癌相关性的研究随之开展。许多证据表明,DNA过甲基化是乳腺癌发生发展过程中的常见现象,且往往意味着乳腺癌关键基因表达的缺失,主要包括细胞周期控制基因、肿瘤易感性基因、肿瘤代谢酶基因和细胞粘附分子基因等。van Hoesel等认为启动子甲基化是乳腺癌发生的早期事件,具有细胞与组织异质性,甲基化表型可早于肿瘤恶变的出现,MINT17, MINT31, RARbeta2, RASSF1A基因甲基化可用于早期诊断及预测肿瘤的恶性程度。Xu等发现BRCA1基因甲基化的三阴乳腺癌患者预后好,且可用于预测此类患者的化疗敏感性。Aran和Hellman分析了转录增强子DNA甲基化与乳腺癌易感性的相关性。基因启动子异常甲基化不仅是多种癌症发生发展过程中重要的表观遗传改变,而且在标本量不大的情况下,比起蛋白质和RNA, DNA甲基化更适合作为早期诊断的敏感生物学指标。由于DNA甲基化在不同肿瘤组织中的高度特异性,检测癌症患者体液中基因异常甲基化越来越受关注。Xu等收集298例乳腺癌患者和612例健康女性外周血标本,检测其甲基化情况,随访5年后发现外周血基因甲基化预测患乳腺癌风险的精确度较Gail模型(65.8%vs.56.0%)和GWASs (65.8%vs.58.8%)均高,认为外周血基因甲基化谱有望成为预测乳腺癌风险的有效指标。
目前虽然在乳腺癌的异常甲基化相关基因研究方面取得了一些进展,但大部分研究只是针对几个或一群基因作为候选基因研究方法。迄今,尚无学者对乳腺癌的异常甲基化在全基因组层面上进行直接、全面系统分析。乳腺癌甲基化谱的建立不仅有助于全面揭示乳腺癌发生的分子机制,更重要的是可为乳腺肿瘤的诊断、治疗、病情监控及预后等提供非常有价值的线索及可靠的科学依据。本研究采用最新发展的全基因组高密度甲基化芯片技术,对乳腺癌组织和正常乳腺组织中异常甲基化的基因进行了初步筛选检测。通过本研究筛选出乳腺癌异常甲基化的基因,初步为乳腺癌甲基化谱的建立奠定了基础,同时也为寻找乳腺癌的诊断、治疗和预后等分子标志物提供可能的线索,而且进一步寻找乳腺癌发生发展的分子机制并有针对性的加以功能验证是我们关注的重点。以下将分成三个方面进行详细介绍。
第一部分Infinium Human Methylation450芯片的乳腺癌甲基化差异性分析
目的：应用高密度基因芯片(450K Infinium Methylation BeadChip)技术分析人类乳腺癌病变组织与正常乳腺组织全基因组的DNA甲基化基因差异水平。
材料与方法：收集6份乳腺癌标本和6份正常乳腺组织标本,提取组织DNA,行重亚硫酸盐转化,与Illumina HD450K Infinium Methylation BeadChip芯片杂交,检测450,000多个甲基化位点,全面覆盖了96%的CpG岛,通过cluster聚类分析和显著性检验分析乳腺癌病变组织与正常乳腺组织中的DNA甲基化水平差异情况,对Delta Data进行筛选统计。结果：乳腺癌病变组织与正常乳腺组织间共发现3268个基因存在DNA甲基化水平差异(Diff Score值50,P<0.00001),甲基化程度升高基因1926个,甲基化程度降低基因1342个。两组全部差异基因的平均甲基化水平实验组较正常对照组高。异常甲基化基因在染色体分布比较均匀,位于1号染色体的基因占14%,位于3号染色体基因占8%。对获得的差异基因进行聚类分析,结果提示：乳腺癌DNA甲基化差异主要集中在细胞粘附分子、细胞信号通路及其能量传导、细胞周期、细胞凋亡相关、细胞周期蛋白类相关基因。这些基因的DNA甲基化改变可能是导致乳腺癌发生发展的原因之一。结论：应用全基因组高密度甲基化芯片对人类乳腺癌病变组织与正常乳腺组织进行的系统研究表明：乳腺癌患者与正常妇女间的基因存在不同的甲基化类型,这些DNA甲基化差异的发现,为进一步研究乳腺癌发生发展的分子机理及其防治新方法的研究奠定了基础。因此,根据显著性检验分析结果,对存在甲基化显著性差异的基因进行进一步的验证,了解这些基因在乳腺癌发生发展过程中的作用。
第二部分RARβ基因甲基化可用于判断淋巴结转移乳腺癌患者的预后
目的：视黄醇受体(Retinoic acid receptor, RAR)基因作用于恶性肿瘤细胞系的反转录过程,抑制肿瘤细胞的增值。RAR与乳腺癌有着非常密切的关系,其中RARβ基因作为乳腺癌抑制因子受到广泛的关注,如果失去了RARβ基因的调节作用就会诱发恶性肿瘤的发生。RARβ基因坐落于3p24上,该区域存在着乳腺癌杂合性丢失的高概率(45%)。但是,LOH不能作为一个独立的因素抑制RARβ基因的表达,因此通过其进一步的研究证明,RARβ基因启动子5’端区域的甲基化与乳腺癌发生具有着重大的关系。本研究的目的是探讨RARβ基因的DNA甲基化在乳腺癌发生发展过程中的意义,为阐明其在乳腺癌的分子机制奠定基础。材料与方法：收集原发性浸润性乳腺癌及其相应的癌旁组织192例,提取组织DNA,行重亚硫酸盐转化,设计了两对特异性扩增引物,应用甲基化特异性PCR (methylation specific-PCR, MSP)法,检测乳腺癌和正常乳腺组织中RARβ基因的DNA甲基化状态,并结合其临床病理特性和预后进行统计学分析。结果：192例乳腺癌组织中RAR β基因甲基化者50例(26%),而在正常乳腺组织中无一例甲基化,差异有统计学意义(P<0.05)。直径>20mm、Ⅲ期乳腺癌及浸润性非导管型乳腺癌与直径<20mm、Ⅰ/Ⅱ期及导管型乳腺癌相比RARβ基因甲基化率具有显著性差异(P<0.05)。淋巴结转移、ER阴性、PR阴性及erbB2过度表达的癌组织中RAR β基因甲基化率增高,但无统计学意义。RARβ基因甲基化率与患者年龄、倍数性及TP53突变无显著相关性(P>0.05)。再对卡方检验中有意义的变量使用logistic回归分析,结果示乳腺癌分期与组织学分型对RARβ基因甲基化与否的影响具有统计学意义。RARβ基因甲基化的肿瘤ER平均值(Mean±SD值)为119±151fmol/μg、PR平均值(Mean±SD值)为91±154fmol/μg,均低于无RARβ基因甲基化肿瘤ER平均值137±169fmol/μg、PR平均值(Mean±SD值)为132±205fmol/μg,但均未达统计学意义。生存曲线分析显示RARβ基因甲基化的乳腺癌患者平均生存65.53(59.21-71.85)月,低于无RARβ基因甲基化患者的69.06(65.84～72.28)月,然而经Log-rank检验,两组生存率曲线的差别无统计学意义(χ2=1.23,P=0.268)。在淋巴结转移的乳腺癌患者中,RARβ基因甲基化的患者平均生存52.43(40.56～64.30)月,显著低于RARβ基因无甲基化的患者的73.13(65.12～81.14)月,经Log-rank检验,两组生存率曲线的差别有统计学意义(χ2=4.230,P=0.040)。结论：RARβ基因甲基化在乳腺癌的发生、发展中起着重要作用,可能与乳腺癌的侵袭性密切相关,可作为判断淋巴结转移的乳腺癌预后的分子生物学指标。
第三部分乳腺癌患者PTPRO基因异常甲基化的预后价值及其外周血检测的临床意义
目的：编码膜结合受体型蛋白酪氨酸磷酸酶O (Protein tyrosine phosphatase receptor-type O, PTPRO)基因是蛋白酪氨酸磷酸酶PTPs家族成员之一,是细胞增殖、分化、代谢、细胞与细胞间通讯、基因转录以及细胞存活等重要信号通路的媒介,参与细胞生长、分化、分裂周期、癌基因转化等多种过程,是新近发现的一个潜在抑癌基因,其在癌症发生机制中的作用是目前研究的热点。另外,检测肿瘤患者外周血中肿瘤相关标志物亦是当前肿瘤研究的热点之一,恶性肿瘤患者外周血中存在游离的肿瘤相关DNA已引起肿瘤学界的极大关注,人们曾在多种肿瘤患者外周血中发现V原发肿瘤相同的DNA变异。本研究以PTPRO基因启动子甲基化作为潜在肿瘤标志物,探讨乳腺癌患者外周血中游离的肿瘤相关DNA及肿瘤组织与临床病理参数包括预后的相关性。材料与方法：采用甲基化特异性PCR法及半定量反转录聚合酶链式反应(reverse transcription PCR, RT-PCR)法,检测乳腺癌组织、癌旁正常腺体组织、乳腺癌细胞株及外周血中游离DNA中PTPRO基因启动子甲基化状况及其表达水平,并结合其临床病理特性进行分析。乳腺癌细胞株培养及去甲基化实验,验证PTPRO基因启动子甲基化与表达水平的相关性。结果：98例乳腺癌组织中PTPRO基因启动子的甲基化率为55%(54/98),与其相应外周血DNA中PTPRO基因启动子的甲基化率为34%(33/98),而对照组正常乳腺组织中无1例有PTPRO基因启动子的甲基化。肿瘤组织未检测到甲基化的患者及健康人血浆中均未检测到该基因甲基化变异。外周血中PTPRO基因启动子的甲基化与肿瘤组织的该基因的甲基化状况显著相关(c=0.435,P=0.000)。发病年龄≥46岁的乳腺癌患者与年龄<46岁的患者PTPRO基因甲基化率有显著性差异(χ2=4.178,P=0.041)。分期晚(χ2=8.616,P=0.003)、低分化级别(χ2=5.139,P=0.023)、淋巴结阳性转移(χ2=6.273,P=0.012)及HER2过度表达(χ2=12.124,P=0.0005)的乳腺癌与分期早、中-高分化、淋巴结无转移及HER2阴性的患者相比肿瘤组织中PTPRO基因甲基化率具有显著性差异。再对卡方检验中有意义的变量使用logistic回归分析,结果示乳腺癌分期与HER2过度表达对PTPRO基因甲基化与否的影响具有统计学意义。ER阴性及PR阴性的癌组织中PTPRO基因甲基化率增高,但未达统计学意义。PTPRO基因甲基化率与患者肿瘤大小、组织学分型及p53肿瘤蛋白(tumor protein53,TP53)突变无显著相关性(P>0.05)。而乳腺癌患者外周血中PTPRO基因甲基化率在HER2过度表达者显著增高(χ2=4.899,P=0.027),与其他临床病理学特征无显著相关性。单变量分析显示PTPRO基因甲基化的乳腺癌患者预后差,且具有统计学意义(χ2=17.240,p=0.000)。在ER+组、PR+组和HER2+组,亦有类似的结果(分别为χ2=12.844,p=0.000:χ2=7.195,p=0.007和χ2=8.603,p=0.003).经多变量分析显示PTPRO基因甲基化可作为乳腺癌患者一个独立的预测预后的分子生物学指标(χ2=5.506；p=0.019)。另外,对PTPRO基因甲基化且表达抑制的乳腺癌细胞株用5-氮杂-2-脱氧胞苷(5-Aza-CdR)进行去甲基化实验,发现PTPRO基因再表达。因此,这些数据显示PTPRO甲基化导致基因失表达。结论：PTPRO基因启动子甲基化在乳腺癌的发生、发展中起着重要作用,与乳腺癌的预后相关,可作为一个独立的预后生物学指标,本研究亦首次报道了PTPRO基因甲基化作为乳腺癌临床诊断的新颖的无创性分子生物学指标的可能性。
总结：
本研究采用最新发展的全基因组高密度甲基化芯片技术,全面覆盖了96%的CpG岛,450,000多个甲基化位点,检测乳腺癌组织和正常乳腺组织,通过cluster聚类分析和显著性检验分析乳腺癌差异表达基因的甲基化差异情况,对异常甲基化的基因进行了初步筛选,结合其在乳腺癌发展过程中的分子生物学功能研究,抽选了其中两个显著性差异(p值最小)的基因进行临床验证,得出如下主要结论：
1. RARβ基因(p=1.44E-07)甲基化可作为判断淋巴结转移的乳腺癌患者预后的分子生物学指标；
2. PTPRO基因(p=2.405E-06)可作为乳腺癌的一个独立的预后生物学指标,以及作为乳腺癌临床诊断的新颖的无创性分子生物学指标的可能。
因此,通过本实验为乳腺癌甲基化谱的建立初步奠定了基础,如果继续扩大研究,有望开发出乳腺癌筛查血清学标志物试剂盒,并应用于临床：肿瘤预警、早期诊断、监控病情、判断预后等。
Introduction:Introduction:According to the data from International Union of Counter Cancer, breast cancer now strikes more women in the world than any other type of cancer. There are currently about1.2million women worldwide fighting breast cancer per year, accounting for the highest incidence (23%) of the global burden of cancers among women. Breast cancer is the leading cause of cancer deaths among women worldwide, with0.3%-8%increasing rate every year. Breast cancer is becoming more and more common among Chinese women and the main killer for their health, making up32%of all new cases. According to the data reported to the National Cancer Registry Center received from a total of49tumor registries in2006, breast cancer contributed to an estimated23.3/100,000new cases and4.71/100,000deaths, resulting in the highest proportion of female cancer in China. The Gail model (GM) for predicting the absolute risk of invasive breast cancer was the most commonly used method in worldwide. Although the GM has been validated in western populations, its performance in other populations is unclear because of the wide variation in international breast cancer rates. However, receiver operating characteristic analysis estimated a prediction accuracy of about58-59%for the Gail model. Mutations in high penetrance cancer susceptibility genes, such as the BRCA1and BRCA2genes, confer a substantially elevated risk to familial breast cancer, accounting for5-10%of breast cancer cases. Genomewide association studies (GWASs) have identified multiple genetic variants associated with breast cancer. The area under the curve (AUC) estimated for10single nucleotide polymorphism (SNPs) from genome-wide association studies of breast was59.7%. Therefore, the known genetic risk and environmental factors in prediction of breast cancer is limited. Early diagnosis and treatment is the key to improve the survival rate and quality of life in patients with breast cancer. Currently the main means of early diagnosis for breast cancer is self examination and imaging examination. However, there are30%misdiagnosis cases. And even15-25%Stage I and II breast cancer will eventually develop distant metastases. Therefore, to further understand the molecular mechanism of breast carcinomas, search the useful targets for the development of novel therapies and improve systemic adjuvant therapy scientifically, is an important content in breast cancer research. Detection of circulating tumor biomarkers is one of the current hot spots in tumor research. Detection of biomarkers in peripheral blood might be used as potential clinical application for cancer prediction, early diagnosis, disease monitoring and decide prognosis. However, there is no ideal result so far.
Malignant tumor is the accumulation of environmental and genetic factors with multi stage, multi steps and abnormal changes in genes. One of the most important mechanisms is the activation of oncogenes and inactivation of tumor suppressor genes. According to Knudson's hypothesis, a tumor suppressor gene generally requires "two hits" to lose its function, which means both alleles must be inactivated. Two pathways by which suppressors become disabled have been widely studied: intragenic mutations and loss of chromosomal material. With more research appeared, people found that DNA sequence is complete and without any mutation or deletion in some cancer. The "two hit theory" cannot explain the reason for the inactivation of tumor suppressor gene. The fact that the traditional genetics cannot answer this phenomenon results in a new discipline-epigenetics, which corresponds with the genetics. DNA methylation is an important epigenetic modification and regulates the function of genome. DNA methylation occurs mainly in the CpG dinucleotide sites with rich G/C content. CpG islands play a role in regulation of gene expression and have special significance. In vertebrate genomes, DNA methylation occurs as a result of the post-replicative addition of a methyl group to selected cytosines to form an altered nucleotide-5-methyl cytosine residue. Approximately70-80%of CpG sites in the human genome are methylated. High concentrations of unmethylated CpG dinucleotides exist in CpG islands. DNA methylation is a regulatory mechanism used to control gene expression and plays a role in such diverse functions as gene imprinting, X-chromosome inactivation, normal development and repression of gene transcription. Much experimental evidence has documented promoter methylation of CpG islands as the third mechanism of inactivation of tumor suppressor, combined with LOH and mutation, and the only mechanism in some cases. It contributes to the carcinogenesis in many tumors. Yan et al. reported the identification of somatic mutations of DNMT3A by exome sequencing and methyaltion microarray analysis in acute monocytic leukemia. They discovered high mutations in DNMT3A together with gene abnormal methylation, which further prove the importance of promoter methylation in cancer research.
Therefore, more and more studies on the correlation between promoter methylation and breast cancer are carrying out. Evidences indicate that DNA hypermethylation is a common phenomenon in the development of breast cancer. It always results in the unexpression of key genes; including cell cycle regulation genes, tumor susceptibility genes, tumor metabolic enzyme genes and cell adhesion molecule gene etc. van Hoesel et al. demonstrated that promoter methylation is an early event in breast cancer with cell and tissue heterogeneity. It might be earlier than the malignant transformation. MINT17, MINT31, RARbeta2and RASSF1A methylation could be used as biomarkers for early detection and present a predictor of malignant potential. Xu et al. found that triple-negative breast cancer patients with BRCA1-methylated tumors are sensitive to adjuvant chemotherapy and have a favorable survival compared with patients with BRCA1-unmethylated triple-negative tumors. Aran and Hellman analyzed the relationship between DNA methylation of transcriptional enhancers and breast cancer predisposition. Gene promoter methylation is not only an important epigenetic change in the development of many cancers, but a suitable sensitively biomarker for early diagnosis, even with small sample volume compared with the detection of protein and RNA. Since hypermethylated DNA may serve as a potential molecular tumor marker due to its high specificity in differentiating cancer from normal tissues, detection of aberrant methylation of tumor suppressor genes in the bodily fluids of cancer patients is attracting increasing attention. Xu et al. collected peripheral blood samples from298breast cancer patients and612healthy women and examined the methylation status. Receiver operating characteristic analysis estimated a prediction accuracy of65.8%for methylation, compared with56.0%for the Gail model and58.8%for genome-wide association study polymorphisms (GWASs). Ther concluded methylation profiling of blood holds promise for breast cancer detection and risk prediction.
Although some progress has been made from studies on aberrant methylation of related genes in breast cancer, most of the research is aimed at several or a group of candidate genes. So far, there is no direct or comprehensively systematic analysis on aberrant methylation at the whole genome level. The construction of methylation profile of breast cancer not only helps to reveal the molecular mechanism, but more importantly to provide a valuable clue and reliable scientific basis for diagnosis, treatment, disease monitoring and prognosis of breast cancer. This study screened the genes with aberrant methylation of breast cancer and normal breast tissue preliminarily using genome wide high-density methylation microaaray technique. Specific genes with aberrant methylation for breast cancer were selected, which initially laid the foundation for the establishment of methylation profiles in breast cancer and provide possible clues for the diagnosis, treatment and prognosis of breast cancer. The focus of our attention is to further understand the molecular mechanism of breast cancer and verify the functions of specific genes. The following will be divided into three aspects in detail.
Part1Methylation analysis in breast cancer based on50K Infinium Methylation BeadChip
Objective:To analyze the methylation level of human breast cancer tissue and normal breast tissue using high-density DNA methylation chip (450K Infinium Methylation BeadChip). Methods:6breast cancer samples and6normal breast tissues were selected for DNA extraction and bisulfite conversion. Illumina HD450K Infinium Methylation BeadChip was applied to detect450,000methylation sites of the whole human genome, covering96%CpG islands. The cluster analysis and significance test were carried out to analyze the different methylation level between human breast cancer tissue and normal breast tissue. And then, the Delta Data was screened for statistics. Results:3268genes showed significant differences in the degree of methylation between breast cancer tissue and normal breast tissue (Diff Score=50, P<0.00001), which included1926hypermethylation genes and1342hypomethylation genes. The average methylation level of significant different genes is higher in the experimental group compared to the control. The chromosome distribution of aberrant methylation genes is quite even,14%at chromosome1and8%at chromosome3. Cluster analysis of the candidate genes showed that:aberrant methylation genes in breast cancer mainly participated in cell adhesion molecules, signal transduction, cell cycle, apoptosis, cell cycle proteins. These genes may be one of the reasons that leaded to breast cancer. Conclusion:Based on the systemic analyses by high-density genome-wide methylation chips between breast cancer tissue and normal breast tissue, we screened out a batch of abnormal methylation genes related to the process of breast caner. This study lays the foundation for further exploration of the molecular mechanism into breast cancer. According the current results, these aberrant methylation genes need to be further studied so that the roles of them in the process of breast carcinogenesis might be understood in the future.
Part2RARβ Gene Methylation can Predict the Prognosis of Breast Cancer Patients with Nodal Involvement
Objective:Retinoic acid receptor (RAR) gene plays a role in the process of reverse transcription and inhibits the cell proliferation in malignant tumor. RARβ gene was considered as a tumor suppressor gene and to be significantly associated with breast cancer, which causes the great interests in recent research. Carcinomas will develop if without the regulation of RARβ gene. RARβ gene locates at3p24. There is high loss of heterozygosity (LOH) rate (45%) in this area. However, LOH couldn't be the independent factor to suppress the expression of RARβ. Therefore, further researches proved that RARP methylation was importantly correlated with breast cancer. The purpose of this study is to determine the roles of RARβ gene in the occurrence and development of breast cancer and provide the foundation for further exploration of the molecular mechanism between breast cancer and RARβ methylation. Methods:192primary invasive breast tumor and the corresponding normal tissues were colleted for DNA extraction and bisulfite conversion. Two sets of specific primers for methylation and unmethylation were decided for PCR. We screened the primary human breast tumors and normal breast tissues for RARβ gene promoter mehtylation using methylation specify PCR (MSP), and the results were analyzed with corresponding clinical pathological data. Results:The frequency of RARβ gene mehtylation among192cases was26%(50/192), however no RARβ gene mehtylation was found in normal breast tissues. The difference was significant (P<0.05). RARβ methylation were associated with tumor histological type, differentiation and tumor size (P<0.05). Patients with lymph node metastasis, ER(-), PR (-) and erbB2amplified had more RARβ methylation, but not significant. No significant association was found between aberrant methylation of RARβ gene and patient age, ploidy and TP53mutation (P>0.05). Average ER value was significantly higher in patients with RARβ methylation (Mean±D=119±151fmol/μg), compared with those with RARβ unmethylation (Mean±SD=137±169fmol/μg). Average PR value was significantly higher in patients with RARP methylation (Mean±SD=91±154fmol/μg), compared with those with RARβ unmethylation (Mean±SD=132±205fmol/μg), but not significant. Kaplan-Meier survival analysis revealed that breast cancer patients with RARβ unmethylation had longer survival of69.06(65.84～72.28) months, compared with65.53(59.21～71.85) months for those with RARβ methylation. However, it was not significant after Long-rank test (χ2=1.23, P=0.268). Then with the subgroup analysis, we found that patients with RARβ methylation [52.43(40.56～64.30)] showed significantly worse survival compared with those with RARβ unmethylation [73.13(65.12～81.14)] in nodal involvement groups (χ2=4.230, P=0.040). Conclusion:RARβ gene promoter mehtylation may play an important role in the carcinogenesis and development of breast cancer. The association with poor differentiation, large tumor size and poor survival indicates that RARβ methylation could predict the prognosis of breast cancer patients with nodal involvement.
Part3Aberrant PTPRO gene methylation predicts clinical outcome in breast cancer patients and its detection in peripheral blood
Objective:Protein tyrosine phosphatase receptor-type O (PTPRO) gene is a member of the family of receptor-type protein tyrosine phosphatases (PTPs). It is an important signaling medium that can affect various cellular processes, including proliferation, differentiation, metabolism, communication, transcription, survival, contact inhibition, cell cycle and oncogene transformation. PTPRO has been described as a new potential tumor suppressor gene in many recent hot researches on carcinogenesis. Since free tumor DNA may exist in the peripheral blood of malignant tumor patients, detection of potential tumor makers in peripheral blood is one of the hot spots in tumor research currently. Recent studies reported that aberrant DNA changes in peripheral blood were associated with those in primary tumors The purpose of this study is to determine the roles of PTPRO gene methylation as a potential biomarker in the detection of breast cancer. And its association with clincopathologic features including prognosis. Methods:We screened the primary human breast tumors, normal breast tissues and peripheral blood for PTPRO gene promoter mehtylation and its expression using methylation specify PCR (MSP) and reverse transcription PCR (RT-PCR). And the results were analyzed with corresponding clinical pathological data. Cell culture and treatment with5-azacytidine was used to analyze the relationship between PTPRO gene mehtylation and its expression. Results:The frequency of PTPRO gene mehtylation among98breast cancer tissues was55%(54/98), and34%(33/98) in peripheral blood, however no PTPRO gene mehtylation was found in normal breast tissues. PTPRO gene methylation in peripheral blood was significantly correlated to that in tumor tissue (c=0.435, P=0.000). PTPRO methylation were associated with patient age (χ2=4.178, P=0.041), tumor stage (χ2=8.616, P=0.003), histological grade (χ2=5.139, P=0.023), lymph node metastasis (χ2=6.273, P=0.012) and erbB2amplified(χ2=12.124, P=0.0005). Patients with ER(-)and PR(-)had more PTPRO methylation, but not significant. No aberrant methylation of PTPRO gene was found in the plasma samples from healthy control and the patients without gene methylation in tumor tissues. In univariate analysis, PTPRO methylation was associated with significantly worse cancer-specific survival in the overall tumor group（χ2=17.240, p=0.000) Subgroup analysis revealed that PTPRO methylation also showed significant prognostic value within the ER+(χ2=12.844, p=0.000), PR+(χ2=7.195, p=0.007) and HER2amplified (χ2=8.603, p=0.003) patient groups. Multivariate analysis showed that PTPRO methylation was an independent factor for worse survival in the overall patient group (χ2=5.506; p=0.019). In addition, demethylation by5-azacytidine treatment led to gene reactivation in PTPRO-methylated and-silenced breast cancer cell lines. Our data suggests that PTPRO methylation is responsible for its inactivation. Conclusion:PTPRO gene promoter mehtylation may play an important role in the carcinogenesis and development of breast cancer. It could be used to predict the prognosis of breast cancer patients. Further, this is the first report of methylated PTPRO as a noninvasive tumor biomarker in peripheral blood of breast tumor patients for detection and disease monitoring.
Conclusion:
The new high-density DNA methylation chip was applied to detect450,000methylation sites of the whole human genome, covering96%CpG islands. The cluster analysis and significance test were carried out to analyze the different methylation level between human breast cancer tissue and normal breast tissue. And then, the Delta Data was screened for statistics. Specific genes with aberrant methylation for breast cancer were selected. Combined with study on the molecular functions during the development of breast cancer, the two significant genes were sletected for further clinical verification. And the conclusion as following:
1. RARβ methylation could predict the prognosis of breast cancer patients with nodal involvement.
2. PTPRO gene promoter mehtylation could be used to predict the prognosis of breast cancer patients. This is the first report of methylated PTPRO as a noninvasive tumor biomarker in peripheral blood of breast tumor patients for detection and disease monitoring.
Therefore, this study initially laid the foundation for the establishment of methylation profiles in breast cancer and provide possible clues for the diagnosis, treatment and prognosis of breast cancer, with the hope of development of screening serum kits for breast cancer.
恶性肿瘤的发生是环境与遗传因素作用下多阶段、多步骤、多基因异常累积的结果,其中最主要的环节是癌基因的激活和抑癌基因的失活。根据经典肿瘤发生的“二次打击理论”,认为抑癌基因的失活有两条途径,即基因内突变和染色质丢失。然而随着研究的深入,人们发现某些恶性肿瘤DNA序列完整,并未有突变、缺失,“二次打击理论”无法解释抑癌基因何以失活。这种用传统遗传学无法解析的现象,催生了与遗传学相对应的另一门新兴学科-一表观遗传学。DNA甲基化是一种重要的遗传外修饰,是表观遗传学的重要组成部分,是调节基因组功能的重要手段。DNA甲基化主要发生在G/C含量丰富的CpG二核苷酸位点(CpG岛),CpG岛对基因的表达起着调控作用,具有特殊的意义。在人类和多数哺乳动物,DNA甲基化仅影响DNA链上鸟嘌呤前的胞嘧啶。正常细胞DNA序列CpG位点约有70-80%发生了甲基化,而大多数CpG岛内CpG位点完全无甲基化。DNA甲基化在基因表达的调控中具有重要作用。近期研究直接分析了基因启动子的CpG岛甲基化,并与LOH或突变分析相结合,证实基因启动子高甲基化是抑癌基因失活的第三种机制,并且在某些情况下是抑癌基因失活的唯一机制,并参与多种肿瘤的发生、发展过程。如Yan等利用外显子测序和甲基化芯片分析发现在急性单核细胞白血病中DNMT3A高突变率,且具有该突变的同时伴随有该基因DNA异常甲基化的出现,进一步验证了启动子甲基化在肿瘤研究中的重要地位。
因此,大量关于启动子异常甲基化与乳腺癌相关性的研究随之开展。许多证据表明,DNA过甲基化是乳腺癌发生发展过程中的常见现象,且往往意味着乳腺癌关键基因表达的缺失,主要包括细胞周期控制基因、肿瘤易感性基因、肿瘤代谢酶基因和细胞粘附分子基因等。van Hoesel等认为启动子甲基化是乳腺癌发生的早期事件,具有细胞与组织异质性,甲基化表型可早于肿瘤恶变的出现,MINT17, MINT31, RARbeta2, RASSF1A基因甲基化可用于早期诊断及预测肿瘤的恶性程度。Xu等发现BRCA1基因甲基化的三阴乳腺癌患者预后好,且可用于预测此类患者的化疗敏感性。Aran和Hellman分析了转录增强子DNA甲基化与乳腺癌易感性的相关性。基因启动子异常甲基化不仅是多种癌症发生发展过程中重要的表观遗传改变,而且在标本量不大的情况下,比起蛋白质和RNA, DNA甲基化更适合作为早期诊断的敏感生物学指标。由于DNA甲基化在不同肿瘤组织中的高度特异性,检测癌症患者体液中基因异常甲基化越来越受关注。Xu等收集298例乳腺癌患者和612例健康女性外周血标本,检测其甲基化情况,随访5年后发现外周血基因甲基化预测患乳腺癌风险的精确度较Gail模型(65.8%vs.56.0%)和GWASs (65.8%vs.58.8%)均高,认为外周血基因甲基化谱有望成为预测乳腺癌风险的有效指标。
目前虽然在乳腺癌的异常甲基化相关基因研究方面取得了一些进展,但大部分研究只是针对几个或一群基因作为候选基因研究方法。迄今,尚无学者对乳腺癌的异常甲基化在全基因组层面上进行直接、全面系统分析。乳腺癌甲基化谱的建立不仅有助于全面揭示乳腺癌发生的分子机制,更重要的是可为乳腺肿瘤的诊断、治疗、病情监控及预后等提供非常有价值的线索及可靠的科学依据。本研究采用最新发展的全基因组高密度甲基化芯片技术,对乳腺癌组织和正常乳腺组织中异常甲基化的基因进行了初步筛选检测。通过本研究筛选出乳腺癌异常甲基化的基因,初步为乳腺癌甲基化谱的建立奠定了基础,同时也为寻找乳腺癌的诊断、治疗和预后等分子标志物提供可能的线索,而且进一步寻找乳腺癌发生发展的分子机制并有针对性的加以功能验证是我们关注的重点。以下将分成三个方面进行详细介绍。
第一部分Infinium Human Methylation450芯片的乳腺癌甲基化差异性分析
目的：应用高密度基因芯片(450K Infinium Methylation BeadChip)技术分析人类乳腺癌病变组织与正常乳腺组织全基因组的DNA甲基化基因差异水平。
材料与方法：收集6份乳腺癌标本和6份正常乳腺组织标本,提取组织DNA,行重亚硫酸盐转化,与Illumina HD450K Infinium Methylation BeadChip芯片杂交,检测450,000多个甲基化位点,全面覆盖了96%的CpG岛,通过cluster聚类分析和显著性检验分析乳腺癌病变组织与正常乳腺组织中的DNA甲基化水平差异情况,对Delta Data进行筛选统计。结果：乳腺癌病变组织与正常乳腺组织间共发现3268个基因存在DNA甲基化水平差异(Diff Score值50,P<0.00001),甲基化程度升高基因1926个,甲基化程度降低基因1342个。两组全部差异基因的平均甲基化水平实验组较正常对照组高。异常甲基化基因在染色体分布比较均匀,位于1号染色体的基因占14%,位于3号染色体基因占8%。对获得的差异基因进行聚类分析,结果提示：乳腺癌DNA甲基化差异主要集中在细胞粘附分子、细胞信号通路及其能量传导、细胞周期、细胞凋亡相关、细胞周期蛋白类相关基因。这些基因的DNA甲基化改变可能是导致乳腺癌发生发展的原因之一。结论：应用全基因组高密度甲基化芯片对人类乳腺癌病变组织与正常乳腺组织进行的系统研究表明：乳腺癌患者与正常妇女间的基因存在不同的甲基化类型,这些DNA甲基化差异的发现,为进一步研究乳腺癌发生发展的分子机理及其防治新方法的研究奠定了基础。因此,根据显著性检验分析结果,对存在甲基化显著性差异的基因进行进一步的验证,了解这些基因在乳腺癌发生发展过程中的作用。
第二部分RARβ基因甲基化可用于判断淋巴结转移乳腺癌患者的预后
目的：视黄醇受体(Retinoic acid receptor, RAR)基因作用于恶性肿瘤细胞系的反转录过程,抑制肿瘤细胞的增值。RAR与乳腺癌有着非常密切的关系,其中RARβ基因作为乳腺癌抑制因子受到广泛的关注,如果失去了RARβ基因的调节作用就会诱发恶性肿瘤的发生。RARβ基因坐落于3p24上,该区域存在着乳腺癌杂合性丢失的高概率(45%)。但是,LOH不能作为一个独立的因素抑制RARβ基因的表达,因此通过其进一步的研究证明,RARβ基因启动子5’端区域的甲基化与乳腺癌发生具有着重大的关系。本研究的目的是探讨RARβ基因的DNA甲基化在乳腺癌发生发展过程中的意义,为阐明其在乳腺癌的分子机制奠定基础。材料与方法：收集原发性浸润性乳腺癌及其相应的癌旁组织192例,提取组织DNA,行重亚硫酸盐转化,设计了两对特异性扩增引物,应用甲基化特异性PCR (methylation specific-PCR, MSP)法,检测乳腺癌和正常乳腺组织中RARβ基因的DNA甲基化状态,并结合其临床病理特性和预后进行统计学分析。结果：192例乳腺癌组织中RAR β基因甲基化者50例(26%),而在正常乳腺组织中无一例甲基化,差异有统计学意义(P<0.05)。直径>20mm、Ⅲ期乳腺癌及浸润性非导管型乳腺癌与直径<20mm、Ⅰ/Ⅱ期及导管型乳腺癌相比RARβ基因甲基化率具有显著性差异(P<0.05)。淋巴结转移、ER阴性、PR阴性及erbB2过度表达的癌组织中RAR β基因甲基化率增高,但无统计学意义。RARβ基因甲基化率与患者年龄、倍数性及TP53突变无显著相关性(P>0.05)。再对卡方检验中有意义的变量使用logistic回归分析,结果示乳腺癌分期与组织学分型对RARβ基因甲基化与否的影响具有统计学意义。RARβ基因甲基化的肿瘤ER平均值(Mean±SD值)为119±151fmol/μg、PR平均值(Mean±SD值)为91±154fmol/μg,均低于无RARβ基因甲基化肿瘤ER平均值137±169fmol/μg、PR平均值(Mean±SD值)为132±205fmol/μg,但均未达统计学意义。生存曲线分析显示RARβ基因甲基化的乳腺癌患者平均生存65.53(59.21-71.85)月,低于无RARβ基因甲基化患者的69.06(65.84～72.28)月,然而经Log-rank检验,两组生存率曲线的差别无统计学意义(χ2=1.23,P=0.268)。在淋巴结转移的乳腺癌患者中,RARβ基因甲基化的患者平均生存52.43(40.56～64.30)月,显著低于RARβ基因无甲基化的患者的73.13(65.12～81.14)月,经Log-rank检验,两组生存率曲线的差别有统计学意义(χ2=4.230,P=0.040)。结论：RARβ基因甲基化在乳腺癌的发生、发展中起着重要作用,可能与乳腺癌的侵袭性密切相关,可作为判断淋巴结转移的乳腺癌预后的分子生物学指标。
第三部分乳腺癌患者PTPRO基因异常甲基化的预后价值及其外周血检测的临床意义
目的：编码膜结合受体型蛋白酪氨酸磷酸酶O (Protein tyrosine phosphatase receptor-type O, PTPRO)基因是蛋白酪氨酸磷酸酶PTPs家族成员之一,是细胞增殖、分化、代谢、细胞与细胞间通讯、基因转录以及细胞存活等重要信号通路的媒介,参与细胞生长、分化、分裂周期、癌基因转化等多种过程,是新近发现的一个潜在抑癌基因,其在癌症发生机制中的作用是目前研究的热点。另外,检测肿瘤患者外周血中肿瘤相关标志物亦是当前肿瘤研究的热点之一,恶性肿瘤患者外周血中存在游离的肿瘤相关DNA已引起肿瘤学界的极大关注,人们曾在多种肿瘤患者外周血中发现V原发肿瘤相同的DNA变异。本研究以PTPRO基因启动子甲基化作为潜在肿瘤标志物,探讨乳腺癌患者外周血中游离的肿瘤相关DNA及肿瘤组织与临床病理参数包括预后的相关性。材料与方法：采用甲基化特异性PCR法及半定量反转录聚合酶链式反应(reverse transcription PCR, RT-PCR)法,检测乳腺癌组织、癌旁正常腺体组织、乳腺癌细胞株及外周血中游离DNA中PTPRO基因启动子甲基化状况及其表达水平,并结合其临床病理特性进行分析。乳腺癌细胞株培养及去甲基化实验,验证PTPRO基因启动子甲基化与表达水平的相关性。结果：98例乳腺癌组织中PTPRO基因启动子的甲基化率为55%(54/98),与其相应外周血DNA中PTPRO基因启动子的甲基化率为34%(33/98),而对照组正常乳腺组织中无1例有PTPRO基因启动子的甲基化。肿瘤组织未检测到甲基化的患者及健康人血浆中均未检测到该基因甲基化变异。外周血中PTPRO基因启动子的甲基化与肿瘤组织的该基因的甲基化状况显著相关(c=0.435,P=0.000)。发病年龄≥46岁的乳腺癌患者与年龄<46岁的患者PTPRO基因甲基化率有显著性差异(χ2=4.178,P=0.041)。分期晚(χ2=8.616,P=0.003)、低分化级别(χ2=5.139,P=0.023)、淋巴结阳性转移(χ2=6.273,P=0.012)及HER2过度表达(χ2=12.124,P=0.0005)的乳腺癌与分期早、中-高分化、淋巴结无转移及HER2阴性的患者相比肿瘤组织中PTPRO基因甲基化率具有显著性差异。再对卡方检验中有意义的变量使用logistic回归分析,结果示乳腺癌分期与HER2过度表达对PTPRO基因甲基化与否的影响具有统计学意义。ER阴性及PR阴性的癌组织中PTPRO基因甲基化率增高,但未达统计学意义。PTPRO基因甲基化率与患者肿瘤大小、组织学分型及p53肿瘤蛋白(tumor protein53,TP53)突变无显著相关性(P>0.05)。而乳腺癌患者外周血中PTPRO基因甲基化率在HER2过度表达者显著增高(χ2=4.899,P=0.027),与其他临床病理学特征无显著相关性。单变量分析显示PTPRO基因甲基化的乳腺癌患者预后差,且具有统计学意义(χ2=17.240,p=0.000)。在ER+组、PR+组和HER2+组,亦有类似的结果(分别为χ2=12.844,p=0.000:χ2=7.195,p=0.007和χ2=8.603,p=0.003).经多变量分析显示PTPRO基因甲基化可作为乳腺癌患者一个独立的预测预后的分子生物学指标(χ2=5.506；p=0.019)。另外,对PTPRO基因甲基化且表达抑制的乳腺癌细胞株用5-氮杂-2-脱氧胞苷(5-Aza-CdR)进行去甲基化实验,发现PTPRO基因再表达。因此,这些数据显示PTPRO甲基化导致基因失表达。结论：PTPRO基因启动子甲基化在乳腺癌的发生、发展中起着重要作用,与乳腺癌的预后相关,可作为一个独立的预后生物学指标,本研究亦首次报道了PTPRO基因甲基化作为乳腺癌临床诊断的新颖的无创性分子生物学指标的可能性。
总结：
本研究采用最新发展的全基因组高密度甲基化芯片技术,全面覆盖了96%的CpG岛,450,000多个甲基化位点,检测乳腺癌组织和正常乳腺组织,通过cluster聚类分析和显著性检验分析乳腺癌差异表达基因的甲基化差异情况,对异常甲基化的基因进行了初步筛选,结合其在乳腺癌发展过程中的分子生物学功能研究,抽选了其中两个显著性差异(p值最小)的基因进行临床验证,得出如下主要结论：
1. RARβ基因(p=1.44E-07)甲基化可作为判断淋巴结转移的乳腺癌患者预后的分子生物学指标；
2. PTPRO基因(p=2.405E-06)可作为乳腺癌的一个独立的预后生物学指标,以及作为乳腺癌临床诊断的新颖的无创性分子生物学指标的可能。
因此,通过本实验为乳腺癌甲基化谱的建立初步奠定了基础,如果继续扩大研究,有望开发出乳腺癌筛查血清学标志物试剂盒,并应用于临床：肿瘤预警、早期诊断、监控病情、判断预后等。
Introduction:Introduction:According to the data from International Union of Counter Cancer, breast cancer now strikes more women in the world than any other type of cancer. There are currently about1.2million women worldwide fighting breast cancer per year, accounting for the highest incidence (23%) of the global burden of cancers among women. Breast cancer is the leading cause of cancer deaths among women worldwide, with0.3%-8%increasing rate every year. Breast cancer is becoming more and more common among Chinese women and the main killer for their health, making up32%of all new cases. According to the data reported to the National Cancer Registry Center received from a total of49tumor registries in2006, breast cancer contributed to an estimated23.3/100,000new cases and4.71/100,000deaths, resulting in the highest proportion of female cancer in China. The Gail model (GM) for predicting the absolute risk of invasive breast cancer was the most commonly used method in worldwide. Although the GM has been validated in western populations, its performance in other populations is unclear because of the wide variation in international breast cancer rates. However, receiver operating characteristic analysis estimated a prediction accuracy of about58-59%for the Gail model. Mutations in high penetrance cancer susceptibility genes, such as the BRCA1and BRCA2genes, confer a substantially elevated risk to familial breast cancer, accounting for5-10%of breast cancer cases. Genomewide association studies (GWASs) have identified multiple genetic variants associated with breast cancer. The area under the curve (AUC) estimated for10single nucleotide polymorphism (SNPs) from genome-wide association studies of breast was59.7%. Therefore, the known genetic risk and environmental factors in prediction of breast cancer is limited. Early diagnosis and treatment is the key to improve the survival rate and quality of life in patients with breast cancer. Currently the main means of early diagnosis for breast cancer is self examination and imaging examination. However, there are30%misdiagnosis cases. And even15-25%Stage I and II breast cancer will eventually develop distant metastases. Therefore, to further understand the molecular mechanism of breast carcinomas, search the useful targets for the development of novel therapies and improve systemic adjuvant therapy scientifically, is an important content in breast cancer research. Detection of circulating tumor biomarkers is one of the current hot spots in tumor research. Detection of biomarkers in peripheral blood might be used as potential clinical application for cancer prediction, early diagnosis, disease monitoring and decide prognosis. However, there is no ideal result so far.
Malignant tumor is the accumulation of environmental and genetic factors with multi stage, multi steps and abnormal changes in genes. One of the most important mechanisms is the activation of oncogenes and inactivation of tumor suppressor genes. According to Knudson's hypothesis, a tumor suppressor gene generally requires "two hits" to lose its function, which means both alleles must be inactivated. Two pathways by which suppressors become disabled have been widely studied: intragenic mutations and loss of chromosomal material. With more research appeared, people found that DNA sequence is complete and without any mutation or deletion in some cancer. The "two hit theory" cannot explain the reason for the inactivation of tumor suppressor gene. The fact that the traditional genetics cannot answer this phenomenon results in a new discipline-epigenetics, which corresponds with the genetics. DNA methylation is an important epigenetic modification and regulates the function of genome. DNA methylation occurs mainly in the CpG dinucleotide sites with rich G/C content. CpG islands play a role in regulation of gene expression and have special significance. In vertebrate genomes, DNA methylation occurs as a result of the post-replicative addition of a methyl group to selected cytosines to form an altered nucleotide-5-methyl cytosine residue. Approximately70-80%of CpG sites in the human genome are methylated. High concentrations of unmethylated CpG dinucleotides exist in CpG islands. DNA methylation is a regulatory mechanism used to control gene expression and plays a role in such diverse functions as gene imprinting, X-chromosome inactivation, normal development and repression of gene transcription. Much experimental evidence has documented promoter methylation of CpG islands as the third mechanism of inactivation of tumor suppressor, combined with LOH and mutation, and the only mechanism in some cases. It contributes to the carcinogenesis in many tumors. Yan et al. reported the identification of somatic mutations of DNMT3A by exome sequencing and methyaltion microarray analysis in acute monocytic leukemia. They discovered high mutations in DNMT3A together with gene abnormal methylation, which further prove the importance of promoter methylation in cancer research.
Therefore, more and more studies on the correlation between promoter methylation and breast cancer are carrying out. Evidences indicate that DNA hypermethylation is a common phenomenon in the development of breast cancer. It always results in the unexpression of key genes; including cell cycle regulation genes, tumor susceptibility genes, tumor metabolic enzyme genes and cell adhesion molecule gene etc. van Hoesel et al. demonstrated that promoter methylation is an early event in breast cancer with cell and tissue heterogeneity. It might be earlier than the malignant transformation. MINT17, MINT31, RARbeta2and RASSF1A methylation could be used as biomarkers for early detection and present a predictor of malignant potential. Xu et al. found that triple-negative breast cancer patients with BRCA1-methylated tumors are sensitive to adjuvant chemotherapy and have a favorable survival compared with patients with BRCA1-unmethylated triple-negative tumors. Aran and Hellman analyzed the relationship between DNA methylation of transcriptional enhancers and breast cancer predisposition. Gene promoter methylation is not only an important epigenetic change in the development of many cancers, but a suitable sensitively biomarker for early diagnosis, even with small sample volume compared with the detection of protein and RNA. Since hypermethylated DNA may serve as a potential molecular tumor marker due to its high specificity in differentiating cancer from normal tissues, detection of aberrant methylation of tumor suppressor genes in the bodily fluids of cancer patients is attracting increasing attention. Xu et al. collected peripheral blood samples from298breast cancer patients and612healthy women and examined the methylation status. Receiver operating characteristic analysis estimated a prediction accuracy of65.8%for methylation, compared with56.0%for the Gail model and58.8%for genome-wide association study polymorphisms (GWASs). Ther concluded methylation profiling of blood holds promise for breast cancer detection and risk prediction.
Although some progress has been made from studies on aberrant methylation of related genes in breast cancer, most of the research is aimed at several or a group of candidate genes. So far, there is no direct or comprehensively systematic analysis on aberrant methylation at the whole genome level. The construction of methylation profile of breast cancer not only helps to reveal the molecular mechanism, but more importantly to provide a valuable clue and reliable scientific basis for diagnosis, treatment, disease monitoring and prognosis of breast cancer. This study screened the genes with aberrant methylation of breast cancer and normal breast tissue preliminarily using genome wide high-density methylation microaaray technique. Specific genes with aberrant methylation for breast cancer were selected, which initially laid the foundation for the establishment of methylation profiles in breast cancer and provide possible clues for the diagnosis, treatment and prognosis of breast cancer. The focus of our attention is to further understand the molecular mechanism of breast cancer and verify the functions of specific genes. The following will be divided into three aspects in detail.
Part1Methylation analysis in breast cancer based on50K Infinium Methylation BeadChip
Objective:To analyze the methylation level of human breast cancer tissue and normal breast tissue using high-density DNA methylation chip (450K Infinium Methylation BeadChip). Methods:6breast cancer samples and6normal breast tissues were selected for DNA extraction and bisulfite conversion. Illumina HD450K Infinium Methylation BeadChip was applied to detect450,000methylation sites of the whole human genome, covering96%CpG islands. The cluster analysis and significance test were carried out to analyze the different methylation level between human breast cancer tissue and normal breast tissue. And then, the Delta Data was screened for statistics. Results:3268genes showed significant differences in the degree of methylation between breast cancer tissue and normal breast tissue (Diff Score=50, P<0.00001), which included1926hypermethylation genes and1342hypomethylation genes. The average methylation level of significant different genes is higher in the experimental group compared to the control. The chromosome distribution of aberrant methylation genes is quite even,14%at chromosome1and8%at chromosome3. Cluster analysis of the candidate genes showed that:aberrant methylation genes in breast cancer mainly participated in cell adhesion molecules, signal transduction, cell cycle, apoptosis, cell cycle proteins. These genes may be one of the reasons that leaded to breast cancer. Conclusion:Based on the systemic analyses by high-density genome-wide methylation chips between breast cancer tissue and normal breast tissue, we screened out a batch of abnormal methylation genes related to the process of breast caner. This study lays the foundation for further exploration of the molecular mechanism into breast cancer. According the current results, these aberrant methylation genes need to be further studied so that the roles of them in the process of breast carcinogenesis might be understood in the future.
Part2RARβ Gene Methylation can Predict the Prognosis of Breast Cancer Patients with Nodal Involvement
Objective:Retinoic acid receptor (RAR) gene plays a role in the process of reverse transcription and inhibits the cell proliferation in malignant tumor. RARβ gene was considered as a tumor suppressor gene and to be significantly associated with breast cancer, which causes the great interests in recent research. Carcinomas will develop if without the regulation of RARβ gene. RARβ gene locates at3p24. There is high loss of heterozygosity (LOH) rate (45%) in this area. However, LOH couldn't be the independent factor to suppress the expression of RARβ. Therefore, further researches proved that RARP methylation was importantly correlated with breast cancer. The purpose of this study is to determine the roles of RARβ gene in the occurrence and development of breast cancer and provide the foundation for further exploration of the molecular mechanism between breast cancer and RARβ methylation. Methods:192primary invasive breast tumor and the corresponding normal tissues were colleted for DNA extraction and bisulfite conversion. Two sets of specific primers for methylation and unmethylation were decided for PCR. We screened the primary human breast tumors and normal breast tissues for RARβ gene promoter mehtylation using methylation specify PCR (MSP), and the results were analyzed with corresponding clinical pathological data. Results:The frequency of RARβ gene mehtylation among192cases was26%(50/192), however no RARβ gene mehtylation was found in normal breast tissues. The difference was significant (P<0.05). RARβ methylation were associated with tumor histological type, differentiation and tumor size (P<0.05). Patients with lymph node metastasis, ER(-), PR (-) and erbB2amplified had more RARβ methylation, but not significant. No significant association was found between aberrant methylation of RARβ gene and patient age, ploidy and TP53mutation (P>0.05). Average ER value was significantly higher in patients with RARβ methylation (Mean±D=119±151fmol/μg), compared with those with RARβ unmethylation (Mean±SD=137±169fmol/μg). Average PR value was significantly higher in patients with RARP methylation (Mean±SD=91±154fmol/μg), compared with those with RARβ unmethylation (Mean±SD=132±205fmol/μg), but not significant. Kaplan-Meier survival analysis revealed that breast cancer patients with RARβ unmethylation had longer survival of69.06(65.84～72.28) months, compared with65.53(59.21～71.85) months for those with RARβ methylation. However, it was not significant after Long-rank test (χ2=1.23, P=0.268). Then with the subgroup analysis, we found that patients with RARβ methylation [52.43(40.56～64.30)] showed significantly worse survival compared with those with RARβ unmethylation [73.13(65.12～81.14)] in nodal involvement groups (χ2=4.230, P=0.040). Conclusion:RARβ gene promoter mehtylation may play an important role in the carcinogenesis and development of breast cancer. The association with poor differentiation, large tumor size and poor survival indicates that RARβ methylation could predict the prognosis of breast cancer patients with nodal involvement.
Part3Aberrant PTPRO gene methylation predicts clinical outcome in breast cancer patients and its detection in peripheral blood
Objective:Protein tyrosine phosphatase receptor-type O (PTPRO) gene is a member of the family of receptor-type protein tyrosine phosphatases (PTPs). It is an important signaling medium that can affect various cellular processes, including proliferation, differentiation, metabolism, communication, transcription, survival, contact inhibition, cell cycle and oncogene transformation. PTPRO has been described as a new potential tumor suppressor gene in many recent hot researches on carcinogenesis. Since free tumor DNA may exist in the peripheral blood of malignant tumor patients, detection of potential tumor makers in peripheral blood is one of the hot spots in tumor research currently. Recent studies reported that aberrant DNA changes in peripheral blood were associated with those in primary tumors The purpose of this study is to determine the roles of PTPRO gene methylation as a potential biomarker in the detection of breast cancer. And its association with clincopathologic features including prognosis. Methods:We screened the primary human breast tumors, normal breast tissues and peripheral blood for PTPRO gene promoter mehtylation and its expression using methylation specify PCR (MSP) and reverse transcription PCR (RT-PCR). And the results were analyzed with corresponding clinical pathological data. Cell culture and treatment with5-azacytidine was used to analyze the relationship between PTPRO gene mehtylation and its expression. Results:The frequency of PTPRO gene mehtylation among98breast cancer tissues was55%(54/98), and34%(33/98) in peripheral blood, however no PTPRO gene mehtylation was found in normal breast tissues. PTPRO gene methylation in peripheral blood was significantly correlated to that in tumor tissue (c=0.435, P=0.000). PTPRO methylation were associated with patient age (χ2=4.178, P=0.041), tumor stage (χ2=8.616, P=0.003), histological grade (χ2=5.139, P=0.023), lymph node metastasis (χ2=6.273, P=0.012) and erbB2amplified(χ2=12.124, P=0.0005). Patients with ER(-)and PR(-)had more PTPRO methylation, but not significant. No aberrant methylation of PTPRO gene was found in the plasma samples from healthy control and the patients without gene methylation in tumor tissues. In univariate analysis, PTPRO methylation was associated with significantly worse cancer-specific survival in the overall tumor group（χ2=17.240, p=0.000) Subgroup analysis revealed that PTPRO methylation also showed significant prognostic value within the ER+(χ2=12.844, p=0.000), PR+(χ2=7.195, p=0.007) and HER2amplified (χ2=8.603, p=0.003) patient groups. Multivariate analysis showed that PTPRO methylation was an independent factor for worse survival in the overall patient group (χ2=5.506; p=0.019). In addition, demethylation by5-azacytidine treatment led to gene reactivation in PTPRO-methylated and-silenced breast cancer cell lines. Our data suggests that PTPRO methylation is responsible for its inactivation. Conclusion:PTPRO gene promoter mehtylation may play an important role in the carcinogenesis and development of breast cancer. It could be used to predict the prognosis of breast cancer patients. Further, this is the first report of methylated PTPRO as a noninvasive tumor biomarker in peripheral blood of breast tumor patients for detection and disease monitoring.
Conclusion:
The new high-density DNA methylation chip was applied to detect450,000methylation sites of the whole human genome, covering96%CpG islands. The cluster analysis and significance test were carried out to analyze the different methylation level between human breast cancer tissue and normal breast tissue. And then, the Delta Data was screened for statistics. Specific genes with aberrant methylation for breast cancer were selected. Combined with study on the molecular functions during the development of breast cancer, the two significant genes were sletected for further clinical verification. And the conclusion as following:
1. RARβ methylation could predict the prognosis of breast cancer patients with nodal involvement.
2. PTPRO gene promoter mehtylation could be used to predict the prognosis of breast cancer patients. This is the first report of methylated PTPRO as a noninvasive tumor biomarker in peripheral blood of breast tumor patients for detection and disease monitoring.
Therefore, this study initially laid the foundation for the establishment of methylation profiles in breast cancer and provide possible clues for the diagnosis, treatment and prognosis of breast cancer, with the hope of development of screening serum kits for breast cancer.
引文
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