颅内原始神经外胚叶肿瘤中TIG1启动子的高甲基化

目的检测颅内原始神经外胚叶肿瘤(primitive neuroectodermal tumor,PNET)中TIG1(tazarotene induced gene-1)基因启动子的甲基化状态。方法使用甲基化特异PCR(MSP)法检测25例原发髓母细胞瘤(medulloblastoma,MB)、9例原发幕上原始神经外胚叶肿瘤(supratentorial primitive neuroectodermal tumor,SPNET)和7株PNET细胞系中TIG1的甲基化水平。用RT-PCR方法检测PNET细胞中TIG1的转录水平。并用去甲基化试剂处理PNET细胞,观察基因转录水平与甲基化状态间的关系。结果原发MB及SPNET中TIG1启动子的异常甲基化率分别为40%(10/25)和44%(4/9)。该基因在PNET中的甲基化异常是肿瘤特异性的。PNET细胞系中均有TIG1高甲基化,并伴有转录水平的表达减少或丢失(7/7)。TIG1的表达水平与启动子甲基化状态呈明显负相关。并且,这种相关性得到去甲基化试剂处理实验的支持。经去甲基化试剂5-aza-2′-deoxycyti-dine处理的全部细胞系中TIG1的表达都得到了恢复。结论大部分PNET细胞系中都有TIG1表达的减少和缺失,且该基因缺失与启动子高甲基化密切相关。同时,TIG1的高甲基化率在原发PNET中也能检测到,提示TIG1可能在颅内PNET的肿瘤发生中起重要作用。     

卵巢癌患者血液中RASSF1A基因甲基化的检测及其意义

目的 探讨循环肿瘤DNA中RASSF1A基因的甲基化及其与卵巢癌的关系。方法 应用甲基化特异性PCR（MSP）方法,对51名正常健康人、51例卵巢癌患者和51例卵巢良性肿瘤患者循环DNARASSF1A基因的甲基化进行了检测。结果 51名正常健康人和51例卵巢良性肿瘤患者循环DNARASSF1A基因甲基化的发生率均为0;51例卵巢癌患者循环肿瘤DNARASSF1A甲基化的发生率为43．1％（22／51,P〈0．05）。RASSF1A甲基化与卵巢癌组织学类型无明显相关性（P〉0.05）。临床Ⅰ期和Ⅱ期循环肿瘤DNARASSF1A基因甲基化的发生率明显低于临床Ⅲ期和Ⅳ期（P〈0．05）;高和中分化组RASSF1A基因甲基化的发生率低于低分化组（P〈0．05）。结论 RASSF1A基因甲基化在卵巢癌的发生和发展过程中起重要作用。卵巢癌患者的循环肿瘤DNA中可以检测到RASSF1A基因的甲基化,与卵巢癌的临床分期和组织学分级有关。循环肿瘤DNA中RASSF1A甲基化的检测有助于卵巢癌的诊断和预后判定。     

RASSF1A及其在肿瘤发生中的作用

RASSF1A（Ras-assotiation domain family 1 A）基因是一个新型抑癌基因。目前的研究已经在许多肿瘤中发现了这个基因的失活。虽然这个基因的失活可因基因缺失或突变引起，但最常见的原因还是该基因的启动子区甲基化紊乱。这种表形遗传学改变被证实是肿瘤形成的一个早期事件，是肿瘤形成的主要因素之一。RASSF1A有多个结构域，被认为是一种潜在的Ras癌蛋白效应分子，能与活化的Ras结合，调节凋亡及细胞周期信号通路，在细胞的凋亡、增殖、分化及维持细胞的稳定中发挥多种生物学效应，与肿瘤的发生发展密切相关。     

FLI-1在小圆细胞肿瘤鉴别诊断中的应用

目的研究FLI-1和CD99在小圆细胞肿瘤中的表达情况,探讨二者在小圆细胞肿瘤鉴别诊断中的应用价值。方法对46例小圆细胞肿瘤进行FLI-1和CD99免疫组化标记,并结合临床与病理组织学进行对比研究。结果FLI-1在Ewing肉瘤／外周原始神经外胚叶肿瘤（EWS／PNET）中阳性表达率为90．5％（19／21）,在分化差的滑膜肉瘤、横纹肌肉瘤分别为14．3％（1／7）、22．2％（2／9）,而在嗅神经母细胞瘤和间叶软骨肉瘤均无表达。CD99在EWS／PNET中阳性表达率为95．2％（20／21）,在分化差的滑膜肉瘤、横纹肌肉瘤、嗅神经母细胞瘤和间叶软骨肉瘤分别为71．4％（5／7）、66．7％（6／9）、75．0％（3／4）和60％（3／5）。FLI-1标记在EWS／PNET的敏感性为90．5％,特异性为88％;而CD99在EWS／PNET的敏感性为95．0％,特异性为32％。FLI-1在EWS／PNET中的特异性明显高于CD99（P〈0．05）。结论FLI-1在EWS／PNET诊断中的价值优于CD99,并且可用于小圆细胞肿瘤的鉴别诊断。     

肺癌患者RASSF1A启动子甲基化状态及其基因表达水平

目的探讨肺癌RAS相关区域家族1A(RASSF1A)启动子CpG岛甲基化状态和基因表达水平与肺癌发生的关系。方法用甲基化特异性PCR检测RASSF1A启动子CpG岛甲基化状态,实时定量PCR检测RASSF1A mRNA的表达水平。结果在肺癌组织中RASSF1A启动子甲基化频率为53.33%(24/45),在正常肺组织中发生甲基化的频率为13.04%(3/23)(P<0.05)。正常肺组织中RASSF1A mRNA全部表达,肺癌组织中表达缺失率为28.89%(13/45),且表达量低于正常肺组织(P<0.05);不同年龄、性别、肿瘤大小、恶性程度、肿瘤分类中其表达量无显著差异;RASSF1A甲基化与其mRNA表达水平下降密切相关。结论肺癌中RASSF1A基因启动子甲基化频率明显升高,其表达普遍下调或缺失,提示RASSF1A启动子甲基化在肺癌发生、发展中起一定作用。     

食管鳞癌分泌素-3A1基因启动子甲基化异常

[目的]研究具有抗癌活性的细胞因子分泌素-3A1基因启动子超甲基化所至该基因表达抑制在我国食管鳞癌发生中的作用。[方法] 用RT-PCR 方法检测分泌素-3A1基因在12个食管鳞癌细胞系的表达,并用甲基化特异PCR技术（methylation specific PCR, MSP）检测上述细胞系及37例来自我国河南的原发性食管鳞癌标本分泌素-3A1基因启动子甲基化状态,通过5-aza-dc处理使培养细胞去甲基化,RT-PCR技术检测处理后该基因在Kyse30的重新表达。[结果] 该基因仅在Kyse410、Kyes520和TE8等3个细胞系有表达,5-aza-dc处理可使Kyse30重新表达分泌素-3A1。此外,在18/37（48%）例原发性食管鳞癌细胞有分泌素-3A1基因启动子区域的超甲基化。[结论] 启动子超甲基化造成分泌素-3A1基因在上述食管鳞癌细胞系表达抑制,原发性食管鳞癌细胞分泌素-3A1基因启动子超甲基化提示该基因表达抑制可能与食管鳞癌发生有关。     

p16、RASSF1A在肝细胞性肝癌患者肿瘤组织及血浆中异常甲基化的检测及临床意义

目的探讨原发性肝细胞性肝癌(HCC)患者肿瘤组织及血浆中p16及RASSF1A启动子区的甲基化状态及其在HCC早期无创诊断中的意义。方法采用甲基化特异性PCR技术检测60例HCC患者肿瘤组织、癌旁组织、血浆及60例正常肝脏患者肝组织及血浆中p16、RASSF1A基因启动子区域的甲基化状态,分析其与肝癌患者临床病理参数之间的关系。结果 60例HCC患者血浆、肿瘤组织及癌旁中p16基因甲基化率分别为68.3%(41/60)、63.3%(38/60)和41.7%(25/60);RASSF1A基因异常甲基化检出率分别为73.3%(44/60)、70.0%(42/60)和36.7%(22/60);60例正常肝脏患者肝组织及血浆p16、RASSF1A未检测到基因启动子区域的甲基化,差异有统计学意义(P=0.000)。HCC患者外周血浆和癌组织中p16、RASSF1A基因的甲基化率与患者年龄、性别、AFP、有无肝炎病毒感染(HBV)、有无肝硬化、Child分级、肿瘤个数、包膜完整与否、有无癌栓、是否复发、病理分级、肿瘤分期无统计学相关性。结论 HCC患者肿瘤组织及血浆DNA中可检测到RASSF1A基因和p16基因的甲基化,外周血p16基因和RASSF1A基因的甲基化检测对肝癌筛查有重要意义,可能成为HCC新的肿瘤分子标记物。     

髓母细胞瘤8号染色体短臂的杂合性丢失

目的研究髓母细胞瘤8号染色体的遗传学异常,寻找与该肿瘤发病机制有关的杂合性丢失位点。方法通过微卫星分析（microsatellite analysis）方法,应用19个位于8号染色体短臂（8p）上的多态性标记物,检测髓母细胞瘤的杂合性丢失（loss of heterozygosity,LOH）。结果在所检测的23例髓母细胞瘤中,21例为原发肿瘤,2例为复发肿瘤。染色体8p总的LOH比率为51%（124个LOH/243个可分析位点）。我们在8p22-23.2之间发现了一个高比率的共同丢失区,其长度为18.14 cM。结论染色体8p22-23.1上很可能存在重要的抑癌基因,该基因的丢失可能与髓母细胞瘤发病有关。     

miR-449a：WNT亚型髓母细胞瘤潜在的表观遗传学标志物北大核心CSCD

目的筛选可能受甲基化调控的,与髓母细胞瘤（medulloblastoma,MB）WNT分子亚型相关的微小RNA（miRNA）分子标志物。方法采用NanoString技术对45例原发性MB石蜡样本进行了包含22个经典MB亚型特异性基因的mRNA表达水平的检测,从而对肿瘤进行分子亚型鉴定。同时通过miRNA表达芯片检测去甲基化药物处理前后MB细胞系（Daoy、D283、D341）的miRNA表达情况,筛选出可能受甲基化调控的miRNA分子标志物,并用甲基化特异性PCR（MSP）和即时荧光定量PCR方法对其甲基化状态和表达水平分别进行验证。最后,对照分子亚型找到与MB亚型相关的受甲基化调控的miRNA。结果45例MB除1例无法准确划分亚型外,其余44例中4例为WNT组,8例为SHH组,16例为Group3,16例为Group4。miR-449a在去甲基化药物处理前后的MB细胞系中表达水平具有明显差异,生物信息学分析确认了其启动子区CpG岛的存在,MSP方法进一步证明了部分肿瘤受启动子高甲基化的调控,且WNT亚型MB肿瘤样本中发现miR-449a存在相对高表达。结论基于金标准（NanoString技术）对MB肿瘤进行分子亚型鉴定,并发现miR-449a在MB细胞系中受到甲基化调控,其可能为WNT亚型MB的分子标志物。     

髓母细胞瘤10号染色体的杂合性丢失分析

目的探讨髓母细胞瘤的遗传学异常及其发病机制。方法通过微卫星分析（microsatellite analysis）方法,应用7个分别位于10号染色体长臂上PTEN（10q23）和DMBT1（10q25）基因位点的特异性标记物,分析18例髓母细胞瘤的杂合性丢失（loss of heterozygosity,LOH）。结果18例髓母细胞瘤中,位于10q23上的LOH比率为24％（9／37可分析标记）;位于10q25上的LOH比率为47％（9／19可分析标记）。结论在髓母细胞瘤中,染色体10q25上高比率的杂合性丢失提示,位于该位点上DMBT1基因的遗传学改变可能在髓母细胞瘤的发病机制中起着重要的作用。     

Hypermethylation of RASSF1A in human and rhesus placentas

The pseudomalignant nature of the placenta prompted us to search for tumor suppressor gene hypermethylation, a phenomenon widely reported in cancer, in the human placenta. Nine tumor suppressor genes were studied. Hypermethylation of the Ras association domain family 1 A (RASSF1A) gene was found in human placentas from all three trimesters of pregnancy but was absent in other fetal tissues. Hypermethylation of Rassf1 was similarly observed in placentas from the rhesus monkey but not the mouse. An inverse relationship between RASSF1A promoter methylation and gene expression was demonstrated by bisulfite sequencing of microdissected placental cells and immunohistochemical staining of placental tissue sections using an anti-RASSF1A antibody. Treatment of choriocarcinoma cell lines, JAR and JEG3, by 5-aza-2'-deoxycytidine and trichostatin A led to reduction in RASSF1A methylation but increased expression. These observations extend the analogy between the primate placenta and malignant tumors to the epigenetic level.     

DNA methylation-dependent silencing of CST6 in human breast cancer cell lines

Cystatin M (CST6) is a candidate breast cancer tumor suppressor that is expressed in normal and premalignant breast epithelium, but not in metastatic breast cancer cell lines. CST6 is subject to epigenetic silencing in MCF-7 breast cancer cells related to methylation of the CpG island that encompasses the CST6 proximal promoter region and exon 1. In the current study, CST6 CpG island methylation and expression status was examined in a panel of breast cancer cell lines. Seven of 12 (58%) cell lines lack detectable expression of CST6 and treatment of these cells with 5-aza-2'-deoxycytidine resulted in a significant increase in CST6 expression, suggesting that the loss of expression may be related to methylation-dependent epigenetic silencing. Bisulfite sequencing of CST6 in a subset of breast cancer cell lines revealed CpG island hypermethylation in CST6-negative cells, and an absence of CpG island methylation in cells that express CST6. The extent of regional methylation was strongly associated with the lack of expression of CST6 among these cell lines. In particular, hypermethylation of the proximal promoter was significantly associated with CST6 gene silencing, and methylation of a number of individual CpGs was found to be statistically correlated with extinction of gene expression. These results establish a strong link between CST6 promoter hypermethylation and loss of CST6 expression in breast cancer cell lines, and suggest that methylation-dependent epigenetic silencing of CST6 may represent an important mechanism for loss of CST6 during breast carcinogenesis in vivo.     

Multipoint methylation analysis indicates a distinctive epigenetic phenotype among testicular germ cell tumors and testicular malignant lymphomas

Hypermethylation of tumor-suppressor genes has been implicated in the pathogenesis of human cancers. Although a growing number of genes showing hypermethylation is being reported in human cancer, methylation profiles of tumor-related genes in testicular neoplasms have not been well elucidated. This study was designed to show the methylation profiles of multiple CpG islands in testicular germ cell tumors (TGCTs) in comparison with those in testicular malignant lymphomas. We studied the methylation status of E-cadherin, CDKN2B, CDKN2A, BRCA1, RB1, VHL, RASSF1A, RARB, and GSTP1 by use of TGCT tissues and testicular malignant lymphoma tissues (25 primary TGCT tissues and three primary testicular lymphoma tissues). Methylation was not observed in E-cadherin, CDKN2B, CDKN2A, BRCA1, RB1, VHL, RASSF1A, RARB, and GSTP1 in any of the TGCT tissues. In contrast, all three (100%) of the testicular lymphoma tissues demonstrated hypermethylation of E-cadherin, RASSF1A, and RARB, but not CDKN2B, CDKN2A, BRCA1, RB1, VHL, and GSTP1. These data demonstrate that a distinctive epigenetic phenotype underlies the TGCTs and testicular lymphomas at the CpG sites of E-cadherin, RASSF1A, and RARB; a distinctive epigenetic phenotype was not observed among seminomatous TGCTs and non-seminomatous TGCTs at the CpG sites examined.     

RASSF1A and NORE1A methylation and BRAFV600E mutations in thyroid tumors

We analyzed RASSF1A and NORE1A methylation and BRAF mutation in 89 thyroid tumors, 42 non-neoplastic thyroid tissues and three thyroid tumor cell lines using polymerase chain reaction (PCR), methylation-specific PCR, Western blotting and DNA sequencing in order to study thyroid tumor pathogenesis and progression. RASSF1A promoter methylation was present in all three thyroid cell lines and in 27/78 (35%) of benign and malignant thyroid tumors. We showed for the first time that there was generally good agreement between RASSF1A methylation status and RASSF1A protein expression. We also examined for the first time NORE1A promoter region methylation in thyroid cell lines and primary tumors and showed that two of three thyroid cell lines were methylated in the NORE1A promoter region, while all primary thyroid tumors analyzed (n=51) were unmethylated. BRAF mutation was present in 38% of papillary thyroid carcinomas (PTC), including 20% of PTC with a follicular variant pattern and 67% of the tall cell variant of PTC. Hyalinizing trabecular tumors (n=23), which had nuclear features similar to PTC, did not have BRAF mutations, indicating that the presence of BRAF mutations can help to separate these two tumor types. Phospho-MEK expression was increased in the NPA cell line, which had a BRAF mutation, supporting the importance of the BRAF pathway alterations in PTC pathogenesis. These results indicate that RASSF1A epigenetic changes are an early event in thyroid tumor pathogenesis and progression and that NORE1A methylation is uncommon in primary thyroid tumors. BRAF mutation occurs later in thyroid tumor progression and is restricted mainly to PTC and anaplastic thyroid carcinoma.     

High frequency of promoter hypermethylation of RASSF1A in tumorous and non-tumourous tissue of breast cancer

AIM: To determine the presence of RASSF1A promoter methylation in tumorous and non-tumorous tissues of breast cancer. METHODS: Methylation-specific PCR was used to detect RASSF1A methylation in DNA extracted from tumorous and paired non-tumorous tissues of 40 breast cancer patients. The associations of RASSF1A hypermethylation with clinicopathological characteristics in tumorous and non-tumorous breast tissues were analysed. RESULTS: RASSF1A promoter hypermethylation was detected in 38 of the 40 breast cancer tissues (95%) and 37 of the paired non-tumorous tissues (92.5%). When compared with the non-tumorous tissues, aberrant methylation was detected to be higher in 24 of the tumorous tissues (60%). The latter was found to be associated with lower histological grade tumours (p=0.048). CONCLUSION: RASSF1A promoter hypermethylation occurred at a high frequency in breast cancer tumorous and non-tumorous tissues; the majority of tumours have a higher level of methylation status when compared with non-tumorous tissues. This supports the notion that RASSF1A methylation is an early and premalignant alteration.