Cancer epigenetics in solid organ tumours: A primer for surgical oncologists

Cancer is initiated through both genetic and epigenetic alterations. The end-effect of such changes to the DNA machinery is a set of uncontrolled mechanisms of cell division, invasion and, eventually, metastasis. Epigenetic changes are now increasingly appreciated as an essential driver to the cancer phenotype. The epigenetic regulation of cancer is complex and not yet fully understood, but application of epigenetics to clinical practice and in cancer research has the potential to improve cancer care. Epigenetics changes do not cause changes in the DNA base-pairs (and, hence, does not alter the genetic code per se) but rather occur through methylation of DNA, by histone modifications, and, through changes to chromatin structure to alter genetic expression. Epigenetic regulators are characterized as writers, readers or erasers by their mechanisms of action. The human epigenome is influenced from cradle to grave, with internal and external life-time exposure influencing the epigenetic marks that may act as modifiers or drivers of carcinogenesis. Preventive and public health strategies may follow from better understanding of the life-time influence of the epigenome. Epigenetics may be used to define risk, to investigate mechanisms of carcinogenesis, to identify biomarkers, and to identify novel therapeutic options. Epigenetic alterations are found across many solid cancers and are increasingly making clinical impact to cancer management. Novel epigenetic drugs may be used for a more tailored and specific response to treatment of cancers. We present a primer on epigenetics for surgical oncologists with examples from colorectal cancer, breast cancer, pancreatic cancer and hepatocellular carcinoma.     

The Relationship between the Methylation of Promoter Regions of Tumor Suppressor Genes PTEN and APC with Endometrial Cancer

Background: Endometrial neoplasms is one of the most typical gynecologic diseases with harmful effects. Promoterhypermethylation is an important mechanism of the inactivation of tumor suppressor genes in endometrial neoplasms.Epigenetic changes of the PTEN and APC genes have shown to be present in various cancers. Therefore, in this study,we have investigated the association between the promoter hypermethylation of PTEN and APC genes with endometrialneoplasms. Methods: For this study, 28 patients with endometrial neoplasms as well as 22 controls were studied.Analysis of the promoter methylation regions of PTEN and APC genes were performed by Methylation-Specific PCR.Results: The frequency of PTEN and APC genes promoter methylation was 28.57% and 17.86% in tumor tissues, and11.54% and 3.85% in blood samples, respectively. We found a significant relationship between blood and tissue inPTEN methylation (p = 0.0353). Additionally, we determined a closely significant difference between normal tissueand tumor tissue of the PTEN gene (p = 0.0787) and blood and tissue samples of the APC gene in methylated promoterregions (p=0.0623). Furthermore, these results suggest that there is no significant relationship between the promotermethylation of PTEN and APC with clinical characteristics. Conclusion: DNA methylation deficiency is a well knownhighlighted factor in tumorigenesis, therefore the promoter hypermethylation of PTEN and APC can be indicated as arisk factor in endometrial neoplasms.     

乳腺癌HOXA4基因甲基化的检测及其临床意义

目的探讨乳腺癌患者HOXA4基因启动子甲基化的情况及其与临床病理特征的相关性。 方法运用NEBNext^? Ultra? RNA Library Prep Kit for Illumina^?进行基因表达芯片测序,运用随机数字表法选择2014年深圳市宝安区妇幼保健院收治的9例乳腺癌患者,分析癌组织与相应癌旁组织异常差异表达的基因。运用Illumina Infinium HD Methylation 450K Assay进行DNA甲基化测序,分析乳腺癌甲基化差异基因。基于肿瘤基因组图谱(The Cancer Genome Atlas,TCGA)数据库信息,分析乳腺癌的差异表达基因和差异甲基化基因。挑选显著高甲基化与低表达的基因,结合生物信息学,确定HOXA4为候选基因。运用随机数字表法收集2014—2017年深圳市宝安区妇幼保健院收治的另外86例乳腺癌患者,采用焦磷酸测序法和RT-PCR,检测乳腺癌组织及其癌旁乳腺组织中HOXA4基因甲基化率和mRNA表达,用Fisher确切概率法分析甲基化率与患者临床病理特征的关系。用Cox比例风险模型进行风险因素的单因素和多因素分析。分别用0、0.5、1、5、10、20 μmol/L的甲基化抑制剂RG108处理乳腺癌MCF-7细胞5 d后,检测HOXA4 mRNA的表达。 结果基因表达数据芯片分析发现在乳腺癌组织中有1 680个显著上调的基因和1 249个下调基因,整体水平上在不同区域乳腺癌患者甲基化水平较癌旁组织高(P均<0.001)。86例乳腺癌组织中HOXA4基因的甲基化率为94% (81/86),其中,30例高甲基化,52例低甲基化;而在对应癌旁组织中,HOXA4基因甲基化率为57%(49/86),其中49例低甲基化,无高甲基化(P<0.001)。有HOXA4甲基化组的癌组织样本中HOXA4 mRNA表达低于无HOXA4甲基化组的癌组织样本(P＝0.003)。HOXA4基因甲基化水平与乳腺癌淋巴结转移、ER表达有关(P＝0.039、0.017)。单因素分析结果显示患者的TNM分期、组织学分级、淋巴结转移及HOXA4甲基化是DFS的危险因素(RR＝4.008,95%CI＝1.296～12.393,P＝0.016;RR＝10.111,95%CI＝2.607～39.217,P＝0.001;RR＝4.588,95%CI＝1.201～17.523,P＝0.026;RR＝1.051,95%CI＝1.007～1.098,P＝0.024)。多因素分析显示组织学分级是乳腺癌患者DFS的独立预后因素(RR＝14.461,95%CI＝2.429～86.100,P＝0.003)。采用不同浓度的RG108来处理MCF-7细胞后,各组HOXA4 mRNA表达比较,差异有统计学意义(χ²＝4.472,P＝0.029)。 结论HOXA4基因启动子甲基化在乳腺癌的发生、发展中起着重要作用,有潜力作为新的分子生物学指标,用于乳腺癌临床诊断。     

三氧化二砷诱导骨髓瘤细胞SOCS-1基因-去甲基化及其对P-STAT3蛋白表达的影响

 目的 探讨三氧化二砷（AS₂O₃) 对多发性骨髓瘤 (MM) 细胞内细胞因子信号转导抑制因子-1（SOCS-1）基因甲基化状态的影响及其对磷酸化的信号转导与转录激活因子-3（P-STAT3）表达的影响。方法 采用甲基特异性PCR法检测AS₂O₃作用前后MM细胞株U266和CZ-1细胞内 SOCS-1基因的甲基化状态,应用蛋白免疫印迹法检测AS₂O₃处理前后细胞内P-STAT3蛋白的表达变化,并采用流式细胞技术检测AS₂O₃作用前后MM细胞增殖和凋亡的变化。结果 MM细胞株内SOCS-1基因存在程度不同的甲基化状态,与对照组相比,AS₂O₃作用后MM细胞内SOCS-1基因甲基化程度明显减弱或消失,P-STAT3蛋白的表达也明显减弱,同时细胞生长受抑,凋亡比率升高。AS₂O₃浓度分别为0、0.5、1.0、2.0 μmol/L时,U266细胞株的总凋亡率分别为0.06%、0.56%、48.96%、61.07%（χ²=9.19,P<0.05）;而CZ-1细胞株的总凋亡率分别为4.20%、40.30%、47.72%、68.49%（χ²=8.96,P<0.05）。结论 AS₂O₃可能通过诱导MM细胞内SOCS-1基因去甲基化作用,进一步抑制细胞增殖信号Janus激酶（JAK）-STAT通路的活化,从而诱导MM细胞的凋亡。     

EID3参与调控人脐带间充质干细胞转分化为类神经干细胞过程的研究

目的研究P300抑制蛋白E1A样转分化抑制蛋白3（EID3）在脐带间充质干细胞（UMSC）转分化为类神经干细胞（uNSCLs）过程中的表达变化及EID3和DNA甲基化转移酶3A（DNMT3A）之间的相互作用关系。 方法（1）通过免疫学实验研究UMSC和uNSCLs细胞表面抗原特点;（2）通过表观遗传学基因检测、蛋白印记实验评价EID3与DNMT3A在UMSC、uNSCLs、神经干细胞（NSCs）中表达量的关系;（3）通过免疫共沉淀实验研究EID3和DNMT3A在UMSC转分化为uNSCLs过程中的相互关系。 结果（1）uNSCLs高表达NSCs的相关mRNA,在Nestin、Psx6、Vimentin、GFAP、Musashi1和NeuroD1基因mRNA的水平上较UMSCs增加了3~13.2倍,差异具有统计学意义（t=9.925、14.089、4.303、8.994、10.122、22.327、1.876;P=0.018、0.0005、0.026、0.0003、0.017、0.002、0.258）;（2）在UMSCs转分化为uNSCLs的过程中,EID3的表达量降低,而DNMT3A则增高,DNMT3A的mRNA水平上在uNSCLs和NSCs中的表达量明显高于UMSCs,差异具有统计学意义（t_uNSCLs=7.453,P_uNSCLs=0.006;t_NSCs=12.924,P_NSCs=0.001）,蛋白水平检测发现,UMSCs转分化为uNSCLs前后,EID3表达降低而DNMT3A则增高,呈相反关系;（3）在uNSCLs细胞中,EID3和DNMT3A共定位于细胞核中,在uNSCLs细胞中,EID3直接结合DNMT3A,并且,HEK293细胞内转染的外源性EID3和DNMT3A同样能相互结合。 结论在UMSCs转分化为uNSCLs细胞的过程中,EID3的表达量逐渐增加并伴随着DNMT3A的表达量逐渐下调,同时两者发生直接结合作用,EID3参与调控了UMSC向uNSCLs的转分化过程。     

Clinical characteristics and outcomes of patients with BRCA1 or RAD51C methylated versus mutated ovarian carcinoma

Objective

In ovarian carcinoma, mutations in homologous recombination DNA repair (HRR) genes, including BRCA1 and RAD51C, are associated with increased survival and specific clinical features. Promoter hypermethylation is another mechanism of reducing gene expression. We assessed whether BRCA1 and RAD51C promoter hypermethylation is associated with similar survival and clinical characteristics.

E-钙黏蛋白基因启动子区甲基化在卵巢子宫内膜异位症中作用的研究

目的　探讨E-钙黏蛋白基因（CDH1）启动子区甲基化状态及其在卵巢子宫内膜异位症中的作用。方法　选择2011年10月—2013年8月河北医科大学第四医院诊治的50例经手术确诊的卵巢子宫内膜异位症患者和50例由于宫颈上皮内瘤变（CIN）Ⅲ级行子宫切除或子宫探查的对照妇女为研究对象,采用甲基化特异性聚合酶链式反应检测卵巢子宫内膜异位症患者在位内膜（在位内膜组）、异位内膜（异位内膜组）和对照妇女子宫内膜组织（对照子宫内膜组）CDH1启动子区甲基化状态;采用反转录聚合酶链式反应（RT-PCR）和流式细胞术检测CDH1启动子区甲基化阳性组织（甲基化阳性组）和甲基化阴性组织（甲基化阴性组）CDH1 mRNA、E-钙黏蛋白（E-cadherin）表达水平。结果　在位内膜组CDH1启动子区甲基化阳性率为26.0%（13/50）、异位内膜组CDH1启动子区甲基化阳性率为32.0%（16/50）、对照子宫内膜组为8.0%（4/50）,在位内膜组和异位内膜组CDH1启动子区甲基化阳性率明显高于对照子宫内膜组（P=0.017、0.003）。甲基化阳性组CDH1 mRNA、E-cadherin表达水平〔（0.4±0.2）、（129.0±17.5）〕明显低于甲基化阴性组〔（0.6±0.1）、（158.0±51.0）〕（P=0.023、0.035）。结论　卵巢子宫内膜异位症患者的在位、异位内膜组织存在着CDH1启动子区异常甲基化,甲基化阳性、阴性组织中CDH1 mRNA和E-cadherin表达水平有明显差异。提示CDH1启动子区的异常甲基化可能与卵巢子宫内膜异位症的发生相关。     

表观遗传异常与宫颈癌

摘要:癌症是一类遗传及表观遗传异常的复杂疾病。宫颈癌由癌前病变发展为侵入性的宫颈癌是由于人乳头瘤病毒（Human Papilloma Virus,HPV）持续感染引起了宿主基因组及表观遗传基因组的改变。表观遗传改变包括DNA甲基化水平改变、miRNA异常表达、癌基因的激活及抑癌基因沉默等。鉴于miRNA可被表观遗传机制调控,因而其在宫颈癌中的异常表达可能是启动子甲基化水平改变引起的。基于以上判断,本文对HPV感染、DNA甲基化水平改变及miRNA异常表达间的关系进行综述,以期揭示表观遗传异常与宫颈癌发生发展之间的关系。