Distinct promotor methylation at tumor suppressive genes in ovarian cancer stromal progenitor cells and ovarian cancer and its clinical implication

Aberrant CpG-island methylation affects ovarian cancer progression. The promotor methylation changes at tumor suppressive genes in ovarian cancer stromal progenitor cells (OCSPCs) and epithelial ovarian cancer (EOC) tissues and their clinical implication remains unexplored. We systemically analyzed the promoter methylation status of 40 tumor suppressor genes (TSGs) associated with cancer in paired epithelial-like and mesenchymal-like OCSPCs and ovarian cancer cells by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The effect of DNA methylation on gene expression was confirmed using qRT-PCR. The differential frequencies of TSGs’ promoter methylation among matched epithelial-like or mesenchymal-like OCSPCs from tissues and ascites and ovarian cancer tissues were further validated in cancer tissues and correlated with clinicopathological features and survival outcomes of patients. According to the promoter methylation frequencies of the 40 TSGs, promoters of RASSF1A were the only significantly hypomethylated in epithelial-like OCSPCs from tissues than those from ascites and bulk tumor cells (0% vs 38% vs 45%, P=0.039 by Fisher’s exact test). The most frequencies at promotor hypermethylation of TSGs in mesenchymal-like OCSPCs from ascites which processed aggressiveness were CDKN2B (73%) followed by CCND2 (45%) and RASSF1A (45%). Forty-three percent (47/110) of RASSF1A and 45% of CCND2 were validated as a frequently hypermethylated gene in an independent set of 110 EOC tissues in contrast to none (0/60) and 12% (10/60) of benign ovarian cysts (both P<0.001). Functional experiments revealed overexpression of CCND2 or CDKN2B in MSc-OCSPCs decreases EMT, invasion, and spheroid formation in EOC, and abolishes DNMT1 and COL6A3 expression. However, for the expected 5-year overall survival (OS) for patients with methylated RASSF1A, CCND2, and CDKN2B, only RASSF1A was significantly worse than those without methylated RASSF1A (56% vs 80%, p=0.022). Taken together, overexpression of CCND2 and CDKN2B decreased the aggressiveness of mesenchymal-like OCSPCs from ascites which may represent a potential therapeutic target for EOC. Promotor hypomethylation at RASSF1A in OCSPCs from EOC tissues and changes to hypermethylation of EOC and OCSPCs from ascites could predict poor survival outcomes for EOC patients compared to without those changes of CCND2 and CDKN2B.     

Methylation Profile of BRCA1, RASSF1A and ER in Vietnamese Women with Ovarian Cancer

DNA methylation is considered a promising biomarkers for diagnosis of cancer in general and of ovariancancer in particular. In our study, we validated the accuracy of methylation specific polymerase chain reaction(MSP) to analyze the methylation pattern of BRCA1, RASSF1A and ER in 59 and 10 Vietnamese patients withepithelial ovarian cancer (EOC) and benign ovarian tumors, respectively. We found methylation of BRCA1,RASSF1A and ER in 11/59 (18.6%), 40/59 (67.8%) and 15/59 (25.4%) of EOC cases, while methylation of BRCA1was only detected in 2/10 (20%) benign ovarian patients. Forty five out of the 59 EOCs (78%) demonstratedmethylation at one or more genes. The methylation frequency of RASSF1A was significantly associated with EOC(p<0.0005). No significant association was observed between methylation status of these genes and the clinicaland pathological parameters of tumors collected from Vietnamese women suffering from ovarian cancer.     

上皮性卵巢肿瘤RASSF1A基因甲基化与蛋白表达的关系及意义

目的:检测上皮性卵巢肿瘤中RASSF1A基因启动子CpG岛的甲基化状态,并探讨基因异常甲基化与蛋白表达的关系及其意义。方法:运用甲基化特异性PCR(MSP)方法对62例上皮性卵巢癌、21例交界性囊腺瘤及30例良性囊腺瘤的RASSF1A基因启动子甲基化状态进行检测,免疫组化S-P法检测上述标本中RASSF1A蛋白表达,并结合肿瘤生物学行为进行分析。结果:上皮性卵巢癌组织、卵巢交界性囊腺瘤组织中RASSF1A基因启动子区甲基化率(58.06%、42.85%)显著高于卵巢良性囊腺瘤组织(13.33%),差异有统计学意义(P<0.05)。RASSF1A基因启动子区甲基化与上皮性卵巢癌的细胞分化程度和临床分期密切相关(P<0.05)而与组织类型和患者年龄及绝经与否无相关性(P>0.05);RASSF1A基因甲基化与其蛋白表达下降一致。结论:卵巢癌组织中存在RASSF1A基因启动子CpG岛的异常甲基化,可能和该蛋白表达缺失的主要原因,是导致该基因失活的重要机制之一。     

上皮性卵巢癌hTERT表达及端粒酶活性的研究

目的探讨人端粒酶反转录酶(hTERT)表达及端粒酶活性与卵巢癌发生、发展的关系,评价其可否作为卵巢癌的肿瘤标志物及预后指标.方法应用RT-PCR及端粒重复扩增方法对43例上皮性卵巢癌、11例良性卵巢肿瘤和卵巢正常组织以及4例交界性卵巢肿瘤共69份标本进行hTERTmRNA及端粒酶活性检测.结果hTERT表达及端粒酶活性率在卵巢癌中分别为83.7%(36/43)及76.7%(33/43),良性肿瘤中均为9%(1/11),在正常卵巢组织和交界性卵巢肿瘤中阴性,其差异在卵巢癌与良性肿瘤及正常组织间均有显著性(P值分别为0.0004及0.0001).hTERT及端粒酶阳性率在某些预后因素如肿瘤类型、组织学分级、临床分期及肿瘤转移之间的差异皆无显著性.结论卵巢癌中存在高频率的hTERT表达及端粒酶激活,表明hTERT作为端粒酶的催化亚基,在肿瘤细胞端粒酶的激活中起关键作用.hTERT表达及端粒酶活性可望作为有价值的肿瘤标志物而用于卵巢癌的早期诊断,但其预后价值尚有待于进一步研究.     

上皮性卵巢肿瘤RASSF1A基因甲基化与蛋白表达的关系及意义

目的：检测上皮性卵巢肿瘤中RASSF1A基因启动子CpG岛的甲基化状态,并探讨基因异常甲基化与蛋白表达的关系及其意义。方法：运用甲基化特异性PCR（MSP）方法对62例上皮性卵巢癌、21例交界性囊腺瘤及30例良性囊腺瘤的RASSF1A基因启动子甲基化状态进行检测,免疫组化S-P法检测上述标本中RASSF1A蛋白表达,并结合肿瘤生物学行为进行分析。结果：上皮性卵巢癌组织、卵巢交界性囊腺瘤组织中RASSF1A基因启动子区甲基化率（58.06%、42.85%）显著高于卵巢良性囊腺瘤组织（13.33%）,差异有统计学意义（P〈0.05）。RASSF1A基因启动子区甲基化与上皮性卵巢癌的细胞分化程度和临床分期密切相关（P〈0.05）而与组织类型和患者年龄及绝经与否无相关性（P〉0.05）;RASSF1A基因甲基化与其蛋白表达下降一致。结论：卵巢癌组织中存在RASSF1A基因启动子CpG岛的异常甲基化,可能和该蛋白表达缺失的主要原因,是导致该基因失活的重要机制之一。     

诱骗受体3在上皮性卵巢癌组织中的表达及临床意义

目的:诱骗受体3(decoy receptor 3,DcR3)是肿瘤坏死因子受体(TNFR)家族的成员,影响着多种肿瘤的发生发展,本实验研究探讨其与卵巢癌发生发展及预后的关系。方法:应用免疫组织化学SP法检测38例正常卵巢组织、29例卵巢良性肿瘤及86例卵巢上皮性癌组织中DcR3蛋白的表达情况。结果:DcR3蛋白在卵巢癌组织中的表达明显高于卵巢良性肿瘤及正常组织中的表达;DcR3蛋白在卵巢上皮性癌Ⅰ-Ⅱ期表达较Ⅲ-Ⅳ期明显减弱;在高中分化组织中的表达明显低于低分化组织;在淋巴结转移组的表达高于无淋巴结转移组,各组间差异均具有统计学意义（P<0.05）。DcR3蛋白表达越强,患者生存时间越短（P<0.05）。结论:DcR3表达水平与卵巢上皮性癌临床分期、组织分化、肿瘤浸润、转移及预后有关,有可能成为一种卵巢癌肿瘤特异性指标。     

贲门腺癌中RASSF1A基因的甲基化状态及表达

 目的 研究贲门腺癌（gastric cardiac adenocarcinoma,GCA）中RASSF1A基因的甲基化状态及其蛋白表达情况。 方法 分别应用甲基化特异性PCR（MSP）、RT-PCR及免疫组织化学SP法检测贲门癌组织及相应癌旁组织的RASSF1A甲基化情况和mRNA水平及蛋白表达情况。 结果 92例贲门癌组织中有54例发生了甲基化,甲基化率为58.7%,显著高于癌旁正常组织（P<0.01）。Ⅲ期和Ⅳ期贲门癌患者中RASSF1A基因发生甲基化的比率显著高于Ⅰ期和Ⅱ期患者（P<0.05）。92例贲门癌组织中有43例RASSF1A基因蛋白表达阴性,与相应癌旁正常组织相比有显著性差异（P<0.01）。Ⅲ期和Ⅳ期贲门癌RASSF1A基因蛋白表达显著低于Ⅰ期和Ⅱ期患者（P<0.05）。发生甲基化的贲门癌组织中RASSF1A的mRNA水平的表达显著低于未发生甲基化的贲门癌组织（P<0.01）。 结论 RASSF1A基因启动子区发生甲基化导致的基因沉默可能是贲门腺癌发生的机制之一。     

The RASSF gene family members RASSF5, RASSF6 and RASSF7 show frequent DNA methylation in neuroblastoma

ABSTRACT: BACKGROUND: Hypermethylation of promotor CpG islands is a common mechanism that inactivates tumor suppressor genes in cancer. Genes belonging to the RASSF gene family have frequently been reported as epigenetically silenced by promotor methylation in human cancers. Two members of this gene family, RASSF1A and RASSF5A have been reported as methylated in neuroblastoma. Data from our previously performed genome-wide DNA methylation array analysis indicated that other members of the RASSF gene family are targeted by DNA methylation in neuroblastoma. RESULTS: In the current study, we found that several of the RASSF family genes (RASSF2, RASSF4, RASSF5, RASSF6, RASSF7, and RASSF10) to various degrees were methylated in neuroblastoma cell lines and primary tumors. In addition, several of the RASSF family genes showed low or absent mRNA expression in neuroblastoma cell lines. RASSF5 and RASSF6 were to various degrees methylated in a large portion of neuroblastoma tumors and RASSF7 was heavily methylated in most tumors. Further, CpG methylation sites in the CpG islands of some RASSF family members could be used to significantly discriminate between biological subgroups of neuroblastoma tumors. For example, RASSF5 methylation highly correlated to MYCN amplification and INRG stage M. Furthermore, high methylation of RASSF6 was correlated to unfavorable outcome, 1p deletion and MYCN amplification in our tumor material. In conclusion This study shows that several genes belonging to the RASSF gene family are methylated in neuroblastoma. The genes RASSF5, RASSF6 and RASSF7 stand out as the most promising candidate genes for further investigations in neuroblastoma.     

宫颈癌组织中RASSF1A基因启动子甲基化对其表达水平的影响及临床意义

目的 研究宫颈癌组织中RAS相关区域家族1A基因(RASSF1A)启动子甲基化水平和RASSF1A基因mRNA表达水平,分析其与宫颈癌临床病理参数的关系及临床意义.方法 收集40例宫颈癌组织及相应癌旁组织,采用巢式特异性甲基化方法检测RASSF1A基因启动子甲基化水平,采用实时荧光定量PCR(qRT-PCR)检测宫颈癌和癌旁组织中RASSF1A基因mRNA表达水平.结果 宫颈癌组织中RASSF1A基因mRNA表达水平(0.26±0.05)显著低于癌旁组织(0.28±0.03),差异有统计学意义(t=2.27,P=0.026);宫颈癌组织中RASSF1A基因启动子区的甲基化率(0.71%±0.04%)显著高于癌旁组织(0.66%±0.03%),差异有统计学意义(t=6.78,P=0.000);RASSF1A基因mRNA表达水平与病理分化程度(t=3.31,P=0.002)、国际妇产科联合会(FIGO)分期(t=2.13,P=0.040)、淋巴结转移(t=2.56,P=0.015)、浸润深度(t=2.93,P=0.006)有关;RASSF1A基因启动子区的甲基化水平与病理分化程度(t=2.08,P=0.045)、FIGO分期(t=2.66,P=0.011)、淋巴转移(t=2.22,P=0.033)、浸润深度(t=2.12,P=0.041)有关.结论 宫颈癌组织中RASSF1A基因启动子甲基化和RASSF1A基因mRNA的表达水平与宫颈癌的恶性程度相关,RASSF1A基因甲基化水平有望成为宫颈癌转移风险的重要指标.     

MMP7蛋白及mRNA在卵巢上皮性肿瘤中的表达及意义

目的 探讨MMP7在上皮性卵巢癌组织中的表达及临床意义。方法 采用免疫组化方法及原位杂交方法检测10例正常卵巢组织、10例良性上皮性卵巢肿瘤组织和54例上皮性卵巢癌组织中MMP7蛋白的表达。结果 正常卵巢组织及良性卵巢肿瘤组织中,MMP7蛋白不表达;上皮性卵巢癌组织中,MMP7阳性表达率为88.89%。MMP7的表达与上皮性卵巢癌的临床分期和病理分级有关,与淋巴结转移无关。MMP7的mRNA与蛋白表达呈正相关(P＜0.0001)。结论 上皮性卵巢癌组织中,MMP7蛋白高度表达。MMP7蛋白在上皮性卵巢癌的发生过程中发挥着重要的作用,MMP7蛋白及mRNA表达水平可以作为判断卵巢癌预后的指标。     

Expression and promoter methylation of the RASSF1A gene in sporadic breast cancers in Chinese women

The novel tumor suppressor RASSF1A is frequently inactivated during human tumorigenesis by promoter methylation. In this study, we detected the RASSF1A promoter methylation by methylated-specific PCR and investigated RASSF1A gene expression by semi-quantitative RT-PCR and immunohistochemical staining in 36 cases of breast cancer and their adjacent normal tissues in Chinese women. The promoter methylation of the RASSF1A gene was found to be a frequent event in the breast cancers (61.1%). RASSF1A methylation was not found in the matched adjacent normal tissues. The loss frequency of RASSF1A mRNA was 33.3% and that of the RASSF1A protein was 44.4% in breast cancers. RASSF1A mRNA and protein were all expressed in adjacent normal tissues. The mRNA and protein expression level of RASSF1A was significantly lower in breast cancer than in adjacent normal tissue. However, the promoter methylation of the RASSF1A gene in breast cancers were not correlated with clinical parameters, such as ages, histological types, TNM stages and lymph node metastases. Thus, the promoter methylation of RASSF1A was one reason for the low level of RASSF1A mRNA and protein expression and was a frequent event in primary sporadic breast tumorigenesis in Chinese women.     

卵巢上皮性癌组织中长链非编码RNA PURPL的表达及其意义

  目的  检测长链非编码RNA PURPL（p53 upregulated regulator of p53 levels）在卵巢上皮性癌（epithelial ovarian cancer,EOC）组织中的表达情况,探讨其在卵巢癌发生发展中的作用。  方法  选用开放医学数据库lncRNASNP2、GEPIA和Kaplan-Meier Plotter检索PURPL在EOC组织中的表达及其与预后的关系。收集2012年10月至2015年10月郑州大学第一附属医院105例患者的临床病理资料,其中包括正常卵巢组织20例、良性卵巢上皮性肿瘤组织20例、EOC组织65例,采用实时荧光定量PCR检测上述不同卵巢组织中的PURPL表达情况,分析EOC组织中PURPL表达与EOC临床病理指标的关系,Kaplan-Meier法分析PURPL表达对EOC患者生存的影响。  结果  数据库检索显示,EOC组织中PURPL的表达显著高于正常卵巢组织,PURPL表达升高与EOC患者的总生存（overall survival,OS）率和无复发生存期（recurrence-free survival,RFS）缩短相关。实时荧光定量PCR检测显示,晚期EOC组织中的PURPL表达为0.530±0.004,显著高于正常卵巢组织的0.029±0.001、良性卵巢上皮性肿瘤组织的0.135±0.001和早期EOC组织的0.488±0.006的表达（P<0.0001）。临床分期越晚（χ2=10.785,P=0.001）、有淋巴结转移（χ2=4.481,P=0.034）的EOC组织中的PURPL高表达。PURPL表达水平相对较高的EOC患者的OS和RFS,明显短于PURPL表达水平相对较低的患者（P<0.05）。  结论  PURPL高表达提示EOC患者预后不良,可作为EOC预后监测的潜在标记物。      

RASSF1A和p16 转录本在非小细胞肺癌中的表达及启动子区甲基化

 目的 研究RASSF1A和p16基因在国人非小细胞肺癌（NSCLC）组织中的转录及启动子区甲基化情况，探讨其转录失活的机制，为NSCLC的诊断和治疗寻找新的途径。方法 应用半定量RTPCR和甲基化特异性PCR法分析96例NSCLC及远癌正常肺组织中RASSF1A和p16基因mRNA的表达和启动子区甲基化情况。结果 （1）53．12％（51／96）的NSCLC中RASSF1A表达明显下调或缺失；36．46％（35／96）的p16表达下调或缺失，而远癌正常肺组织均表达良好。（2）96例NSCLC中RASSF1A甲基化率48．96％（47／96），该基因表达明显下调或缺失的51例中39例（76．5％）出现甲基化，表达正常的45例中8例（17．8％）出现甲基化，两组对比差异有统计学意义（P〈0．05）；96例NSCLC中33例（34．38％）检测到p16启动予区甲基化，p16基因表达明显下调的35例中20例（57．1％）出现该基因CPG岛的甲基化，而表达正常的61例中13例（21．3％）出现甲基化，两组比较差异显著（P〈0．05）。96例远癌正常肺组织均未检测到此两基因启动子有甲基化。结论 RASSF1A和p16基因mRNA在国人NSCLC中较高比例的表达下调或缺失；甲基化可能是两基因表达失活的主要原因。     

卵巢上皮性恶性肿瘤组织RASSF1A基因启动子区甲基化的研究

目的 探讨RASSF1A基因启动子区异常甲基化与卵巢上皮性恶性肿瘤发生、发展的关系。方法 应用甲基化特异性PCR方法,检测80例卵巢上皮性恶性肿瘤组织RASSF1A基因启动子区异常甲基化。结果 80例卵巢上皮性恶性肿瘤组织中,RASSF1A基因启动子区甲基化的发生率为52．5％,而相应痛旁正常组织中,RASSF1A基因启动子区均未发生甲基化（P〈0．05）。浆液性癌、黏液性癌和内膜样癌中,RASSF1A基因启动子区甲基化的发生率分别为54．2％、52．4％和45．5％,差异尤统计学意义。临床Ⅰ期、Ⅱ期卵巢上皮性恶性肿瘤RASSF1A基因启动子区甲基化的发生率分别为21．4％和16．7％,明显低于临床Ⅲ期（66．7％）和Ⅳ期（77．8％）。高分化组和中分化组RASSFlA基因启动子区甲基化的发牛率分别为34．5％和35．0％,均低于低分化组（80．6％）。结论 卵巢上皮性恶性肿瘤组织中存在RASSF1A基因启动子区的异常甲基化,甲基化与卵巢上皮性恶性肿瘤的临床分期和组织学分级有关。     

Altered expression of β-catenin, E-cadherin, and E-cadherin promoter methylation in epithelial ovarian carcinoma

The aim of the study was to evaluate the immunoexpression of E-cadherin, β-catenin, and Ki-67, as well as the promoter methylation of E-cadherin gene in epithelial ovarian cancer (EOC), as well as to find a possible relationship between the immunoexpression and hypermethylation. Promoter methylation was studied using methylation-specific PCR in 86 malignant cases, 14 low malignant potential (LMP) tumors and 19 benign cystadenomas. Immunohistochemical expression was carried out in 64 malignant cases, 8 LMP tumors, and 11 benign cystadenomas. Immunoexpression of E-cadherin was reduced in EOC, while 100 % expression was seen in LMP tumors and benign cystadenomas. An interesting observation was the nuclear expression of E-cadherin in a high percentage of cancers, which showed a positive correlation with Ki-67. Β-Catenin expression showed heterogeneous localization with increased nuclear localization, which was significantly higher in cases that did not express E-cadherin. Promoter methylation of E-cadherin was 36, 14, and 11 % in EOC, LMP tumors, and benign cystadenomas, respectively. Our results suggest that reduced expression of E-cadherin is associated with promoter methylation of E-cadherin gene, in addition to providing evidence for the aberrant nuclear localization of E-cadherin in EOC.     

卵巢上皮性癌组织中DAPK基因启动子甲基化及其蛋白表达的研究

  目的  研究卵巢上皮性癌组织中DAPK基因启动子甲基化及其蛋白表达在卵巢癌发生发展过程中的作用及意义。  方法  应用甲基化特异性PCR(MSP)检测55例卵巢上皮性癌(恶性组)、25例卵巢交界性上皮性肿瘤(交界组)、30例卵巢良性上皮性肿瘤(良性组)、25例正常卵巢上皮组织(正常组)的石蜡包埋组织中DAPK基因启动子的甲基化情况。应用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶连接(S-P)法检测上述蜡块组织中DAPK蛋白的表达情况。  结果  DAPK启动子在正常组、良性组、交界组、恶性组的甲基化率分别为0(0/25)、6.7%(2/30)、16.0%(4/25)、47.3%(26/55), 差异有统计学意义(P < 0.001), 恶性组的甲基化率高于其他三组; DAPK蛋白在正常组、良性组、交界组、恶性组的阳性表达率分别为96.0%(24/25)、90.0%(27/30)、48.0%(12/25)、30.9%(17/55), 差异有统计学意义(P < 0.001), 恶性组、交界组的阳性表达率均低于正常组和良性组; DAPK基因启动子甲基化和DAPK蛋白表达呈负相关。  结论  DAPK基因启动子甲基化导致基因沉默、失活, 引起蛋白表达下调或缺失, 并参与了卵巢上皮性癌的发生发展。      

Methylation profiles of sporadic ovarian tumors and nonmalignant ovaries from high-risk women.

PURPOSE: The purpose of this research was to examine the DNA methylation profiles of primary sporadic ovarian cancers and ovarian tissues from high-risk women. EXPERIMENTAL DESIGN: We analyzed the DNA methylation status of nine cancer-related genes in 49 primary ovarian tumors, 39 nonmalignant ovarian tissues obtained from 16 women with no known risk and from 23 high-risk women with a strong family history of breast and/or ovarian cancer or BRCA1 germ-line mutations, and 11 ovarian cancer cell lines, by methylation-specific PCR. RESULTS: Our findings are as follows: (a) methylation rates of four of nine genes, RASSF1A (41%), HIC1 (35%), E-cadherin (29%), and APC (18%) were significantly higher in tumors compared with controls. At least one of the four genes was methylated in 76% of the tumors; (b) a low frequency of methylation was present in nonmalignant tissues; (c) no significant differences in methylation frequencies were seen between the nonmalignant ovarian tissues from women at high-risk and those with no known risk of developing ovarian cancer; (d) methylation of the BRCA1 gene was found in 10% of sporadic tumors but in none of the samples from women with a germ-line BRCA1 mutation; and (e) ovarian cancer cell lines showed a similar frequency of methylation to ovarian tumors except for the HIC1 gene. CONCLUSIONS: Our results suggest that aberrant methylation of specific genes, including two not described previously, may be important in ovarian cancer pathogenesis but not in ovaries at risk for cancer development.