人肝细胞癌金属蛋白酶组织抑制因子-3的表达及与其基因启动子甲基化的关系

目的 探讨肝细胞癌的发生和门脉浸润机制。方法 20例肝细胞癌患者,每例在手术后分别取原发瘤、门脉瘤栓及远离肝癌之肝组织。用Western印迹法检测金属蛋白酶组织抑制因子(TIMP)的蛋白表达,用逆转录-聚合酶链反应(RT-PCR)检测TIMP-3 mRNA的表达,用甲基化特异性PCR检测TIMP-3基因启动子的甲基化。结果 远离肝癌之肝组织中均可见TIMP-3蛋白和mRNA的表达,原发瘤和门脉瘤栓组织TIMP-3蛋白和mRNA表达明显降低,其中分别有5例和6例完全丢失。远离肝癌之肝组织均未发现TIMP-3启动子甲基化。原发瘤中有7例、门脉瘤栓组织中有9例出现甲基化。所有有甲基化的肝癌组织,包括原发瘤和门脉瘤栓,有13例TIMP-3 mRNA和蛋白表达完全丢失,6例表达降低。TIMP-3启动子甲基化和肝细胞癌组织学分级无关(P>0.05)。结论 肝细胞癌的发生和门脉浸润与TIMP-3基因和蛋白缺失或降低相关、而TIMP-3启动子甲基化是其基因和蛋白缺失或降低的原因。     

Frequent silencing of DBC1 is by genetic or epigenetic mechanisms in non-small cell lung cancers

Genome-wide screening of DNA copy number aberrations in 27 cell lines derived from non-small cell lung cancers (NSCLCs), using a custom-made comparative genomic hybridization (CGH)-array, identified a homozygous deletion of the deleted in bladder cancer 1 gene (DBC1) in one cell line. Homozygous deletion of DBC1, located at 9q33.1, was also observed in two of 53 primary NSCLC tumors examined. Moreover, 21 of the other 26 cell lines showed complete loss of DBC1 expression, although normal lung tissues express this gene, and treatment with 5-aza-2'-deoxycytidine restored expression of DBC1. Hypermethylation in part of a CpG island around the exon 1 of DBC1 has been reported in urothelial cancers, but the potential association between methylation and expression status was never clarified in that disease. In our experiments, a different part of the same CpG island showed promoter activity in vitro and was frequently methylated in our cell lines and primary tumors of NSCLC, where methylation status correlated inversely with gene expression. Among our primary NSCLC cases, methylation of the DBC1 promoter occurred more frequently in men, elderly patients and smokers than in women, younger patients and nonsmokers respectively, but it was not correlated with tumor stage or histology. Exogenous overexpression of DBC1 in NSCLC cell lines lacking its expression inhibited cell growth. Our results provide the first evidence that DBC1 is a likely tumor suppressor for NSCLC; silencing of the gene through homozygous deletion or methylation of its promoter region may be associated with progression of this disease.     

Silencing of PCDH10 in hepatocellular carcinoma via de novo DNA methylation independent of HBV infection or HBX expression

PCDH10 is a key tumor suppressive gene for nasopharyngeal, esophageal, and other carcinomas with frequent methylation. In this study, we investigated the potential epigenetic modification of the PCDH10 gene by hepatitis B virus × protein (HBx), a pivotal factor in the progression of HBV replication and potential carcinogenesis. PCDH10 expression was found to be down-regulated in 9/13 (69.2 %) of hepatocellular carcinoma (HCC) cell lines. Decreased PCDH10 expression was correlated with the methylation status of the PCDH10 promoter. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza) was sufficient to restore PCDH10 mRNA expression by suppressing PCDH10 promoter methylation in HepG2 cells. Treatment with Trichostatin A alone had no significant effect on PCDH10 expression but enhanced the effect of Aza. PCDH10 methylation was further detected in 76 % (38 of 50) of HCC tissues compared with 40 % (20 of 50) of paired adjacent tissues, with no methylation detected in normal human liver tissues. There were significant correlations between methylation status of PCDH10 and tumor size, serum AFP levels, metastasis or TNM staging (P < 0.05). Moreover, PCDH10 promoter methylation status was not associated with HBV infection in our panel of 50 primary HCC tumors, and transfection with HBX could not alter the status of PCDH10 promoter methylation. Collectively, these observations suggested that the expression of PCDH10 was silenced in HCC via de novo DNA methylation independent of HBV infection or HBX expression, and PCDH10 might form a potentially useful therapeutic target for HCC.     

CDH1基因启动子甲基化在上皮性卵巢癌转移方面的研究

目的探讨CDH1基因启动子甲基化对上皮性卵巢癌转移的影响。方法采用免疫组织化学方法检测38例正常卵巢上皮和80例上皮性卵巢癌组织中E-钙黏附素（E-cadherin）表达;应用甲基化特异的PCR（MSP）检测上述组织中CDH1基因启动子区甲基化;应用5-氮-2＇-脱氧胞苷（5-aza-CdR）使SKOV3细胞去甲基化,观察SKOV3细胞体外侵袭性的改变,并通过RT-PCR检测CDH1基因的改变。结果E-cadherin在正常卵巢组织中表达明显高于上皮性卵巢癌（P〈0．05）。34例CDH1基因启动子区甲基化全部出现在卵巢癌组织中,有淋巴结转移组织中甲基化明显高于无淋巴结转移者（P〈0．05）,而CDH1基因启动子区有甲基化的卵巢癌组织中E-cadherin表达明显降低（P〈0．05）。经5-Aza-CdR处理后的SKOV3细胞体外侵袭性降低（P〈0．01）,CDH1基因的表达明显上调（P〈0．01）。结论E-cadherin表达降低与上皮性卵巢癌转移关系密切,CDH1基因启动子区甲基化可能是导致E-cadherin蛋白表达减低的重要原因之一,因此启动子区甲基化与上皮性卵巢癌转移有关。     

Decreased expression and aberrant methylation of Gadd45G is associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma

The growth arrest DNA damage-inducible gene (Gadd45) family, which is composed of Gadd45A, Gadd45B, and Gadd45G, is involved in DNA damage response and cell growth arrest. The present study was to detect the role of Gadd45 gene family in esophageal cancer and the relationship of Gadd45G methylation to a series of pathological parameters in a large esophageal squamous cell carcinoma (ESCC) sample, in order to elucidate more information on the role of Gadd45 gene family with regard to the pathogenesis of ESCC. Frequent silencing of Gadd45G but not Gadd45A and Gadd45B were found in esophageal cancer cell lines and the silencing of Gadd45G may be reversed by 5-Aza-dC or TSA treatment in Eca109 cell line. The aberrant proximal promoter methylation of Gadd45G induces silencing of Gadd45G expression in Eca109 cell line. Gadd45A mRNA and protein expression in ESCC tumor tissues was significantly different compared to corresponding normal tissues. Decreased mRNA and protein expression of Gadd45G was observed in ESCC tumor tissues and was associated with Gadd45G proximal promoter methylation. Gadd45A or Gadd45B expression was not correlated with ESCC patients survival, while Gadd45G methylation status and protein expression were independently associated with ESCC patients’ survival. These data indicated that Gadd45G may be a functional tumor suppressor and its inactivation through proximal promoter methylation may play an important role in ESCC carcinogenesis and reactivation of Gadd45G gene may has therapeutic potential and may be used as a prognostic marker for ESCC patients.     

乳腺癌中印记基因SLC22A18/TSSC5/BWR1A启动子区甲基化及mRNA表达的研究

目的： 研究印记基因SLC22A18启动子区甲基化对乳腺侵润性导管癌组织中的SLC22A18 mRNA表达的影响，探讨其与临床病理特征之间的关系。方法：实时荧光定量逆转录聚合酶链反应（real-time quantitative RT-PCR）方法检测40例乳腺侵润性导管癌及其癌旁组织中SLC22A18 mRNA的表达，甲基化特异性聚合酶链反应(MSP)检测SLC22A18基因启动子区的甲基化状态。检测DNA甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷（5-aza-2′-deoxycytidine，5-aza-dc）和组蛋白去乙酰化酶抑制剂曲古霉素A（TSA）作用于乳腺癌细胞株MDA-MB-231后，对SLC22A18基因启动子区DNA甲基化和mRNA表达的影响。结果：SLC22A18在40例乳腺侵润性导管癌中mRNA表达量低于癌旁组织（0.12±0.10 vs 0.69±1.05，P＜0.01）；40例乳腺侵润性导管癌及对应癌旁组织中，SLC22A18启动子区的甲基化发生率分别为75％和37.5%，差异有统计学意义（P＜0.01）；在40例乳腺侵润性导管癌组织中，甲基化组SLC22A18 mRNA表达量低于非甲基化组（0.11±0.08 vs 0.24±0.18，P＜0.01）。5-aza-dc和5-aza-dc/TSA能不同程度逆转乳腺癌细胞株MDA-MB-231中SLC22A18基因的甲基化状态，并上调SLC22A18基因表达。结论：SLC22A18基因甲基化与乳腺癌发生有一定的关联，SLC22A18基因表达下调与其启动子区CpG岛异常甲基化相关。     

遗传性弥漫型胃癌家系上皮型钙黏素基因的分析

 摘要：目的 研究在遗传性弥漫型胃癌家系中是否存在上皮型钙黏素基因(CDH1)以及基因表达的异常。 方法 收集符合遗传性弥漫型胃癌(HDGC)诊断标准的1个家系中15例成员的外周血和组织标本。通过免疫组化和Western blot 的方法检测组织标本CDH1蛋白表达; 提取基因组DNA, 通过PCR扩增DNA直接测序检测CDH1基因16个外显子突变。用克隆测序法,鉴定CDH1基因启动子区CpG位点甲基化状况。 结果 先证者和另一胃癌患者(家系2号成员)的癌旁胃粘膜上皮细胞CDH1蛋白表达较正常胃粘膜减弱, 两者肿瘤组织的蛋白表达几乎为阴性。包括先证者在内的11例家系成员第14外显子mRNA水平2377位点存在一个C?T的单核苷酸多态性(single nucleotide polymorphism, SNP), 但未检测到16个外显子的胚系突变。相对于正常胃粘膜, 先证者和家系2号成员的胃癌组织均有CDH1基因启动子的高甲基化, 其瘤旁粘膜也有高甲基化。结论 此HDGC家系中,CDH1基因表达丢失可能和胃癌发生有关, CDH1基因外显子突变不是其肿瘤组织上皮型钙黏素蛋白表达丢失的直接原因, 基因启动子区的甲基化可能是导致其失活的原因之一。     

The effects of estrogen on prolactin gene methylation in normal and neoplastic rat pituitary tissues.

The effects of estrogen treatment on rat prolactin (PRL) gene methylation were analyzed in normal pituitaries and in three transplantable rat pituitary tumors. Northern analysis showed increased PRL mRNA expression in estrogen-treated pituitary and in MtT/F4 and MtT/F-DMBA tumors. Prolactin mRNA amounts in MtT/W15 tumor were decreased by estrogen treatment. There was an inverse relationship between changes in PRL mRNA expression and changes in gene methylation in the coding regions of the PRL gene after estrogen treatment. The amounts of the 4.6-Kb and 1.8-Kb restriction fragments generated by HpaII digestion in pituitary tissues were influenced by estrogen, with an increase in these fragments in normal pituitary, MtT/F4, and MtT/F-DMBA tumors after estrogen treatment. In contrast, the amounts of the 4.6-Kb and 1.8-Kb fragments were decreased in MtT/W15 tumors after estrogen treatment. Most of the internal -CCGG- sites in the PRL gene were methylated in the liver, and the PRL gene was not expressed in the liver. These data suggest that there is a tissue-specific pattern of DNA methylation of the PRL gene and that PRL gene methylation is influenced by estrogen in vivo in normal and tumorous pituitary tissues. These results also suggest that estrogen may influence PRL expression by multiple mechanisms, including changes in the level of DNA methylation.     

EXTL3 promoter methylation down-regulates EXTL3 and heparan sulphate expression in mucinous colorectal cancers

Exostoses like-3 (EXTL3) is a putative tumour suppressor gene but its involvement in colorectal cancer (CRC) is unclear. We have investigated the role of methylation of the EXTL3 promoter as a mechanism for EXTL3 regulation and tested the hypothesis that loss of EXTL3 expression is associated with mucinous differentiation and alteration of glycoprotein expression in CRC cells. The methylation status of the EXTL3 gene promoter was analysed by methylation-specific PCR following bisulphite modification in CRC cell lines and microdissected primary CRC tissues and their corresponding adjacent normal colorectal mucosa. EXTL3 promoter methylation was detected in seven of 11 mucinous CRCs (63.6%) but in none of 26 non-mucinous CRCs examined. EXTL3 promoter methylation was also detected in the normal colonic mucosa of three patients with mucinous CRC but not in the normal colonic mucosa of any patients with non-mucinous CRC. The presence of EXTL3 methylation was significantly associated with the partial loss of HS expression in mucinous CRC lesions. The mucinous CRC cell lines, Colo201 and Colo205, showed EXTL3 promoter methylation and loss of EXTL3 mRNA expression. However 5-aza-2'-deoxycytidine treatment demethylated the EXTL3 gene promoter and restored its mRNA expression. Furthermore, the basal expression of HS in CRC cells was abolished by treatment with EXTL3-siRNA. We conclude that EXTL3 promoter methylation and its related loss of EXTL3 expression are involved in the loss of HS expression in mucinous CRCs.     

Hypermethylation-mediated regulation of CD44 gene expression in human neuroblastoma

The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single-strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44-negative cell lines displayed hypermethylation in both regions, whereas all CD44-expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44-negative cells with the 5-aza-2'-deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation-independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell-specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation-mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers.     

Prognostic CpG Methylation Biomarkers Identified by Methylation Array in Esophageal Squamous Cell Carcinoma Patients

Background: Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with poor prognosis. We aimed to identify a panel of CpG methylation biomarkers for prognosis prediction of ESCC patients.Methods: Illumina''s GoldenGate methylation array, supervised principal components, Kaplan-Meier survival analyses and Cox regression model were conducted on dissected tumor tissues from a training cohort of 40 ESCC patients to identify potential CpG methylation biomarkers. Pyrosequencing quantitative methylation assay were performed to validate prognostic CpG methylation biomarkers in 61 ESCC patients. The correlation between DNA methylation and RNA expression of a validated marker, SOX17, was examined in a validation cohort of 61 ESCC patients.Results: We identified a panel of nine CpG methylation probes located at promoter or exon1 region of eight genes including DDIT3, FES, FLT3, NTRK3, SEPT5, SEPT9, SOX1, and SOX17, for prognosis prediction in ESCC patients. Risk score calculated using the eight-gene panel statistically predicted poor outcome for patients with high risk score. These eight-gene also showed a significantly higher methylation level in tumor tissues than their corresponding normal samples in all patients analyzed. In addition, we also detected an inverse correlation between CpG hypermethylation and the mRNA expression level of SOX17 gene in ESCC patients, indicating that DNA hypermethylation was responsible for decreased expression of SOX17.Conclusions: This study established a proof-of-concept CpG methylation biomarker panel for ESCC prognosis that can be further validated by multiple cohort studies. Functional characterization of the eight prognostic methylation genes in our biomarker panel could help to dissect the mechanism of ESCC tumorigenesis.     

HOXD13 methylation status is a prognostic indicator in breast cancer

Homeobox protein Hox-D13 is encoded by HOXD13 gene which is frequently methylated in cancer and has been recognized as a tumor suppressor in pancreatic cancer. In this study, we examined HOXD13 mRNA expression in 40 pairs of breast cancers and corresponding normal breast tissues. Bisulfite sequencing of HOXD13 promoter was performed in 6 pairs of breast tumors and corresponding normal breast tissues to examine the potential HOXD13 CpG methylated sites. HOXD13 DNA methylation frequency analysis was performed using MethyLight in 196 pairs of breast cancers and corresponding normal breast samples. DNA methylation status and clinico-pathological features were investigated. Kaplan-Meier survival analysis and Cox proportional hazards models were utilized to assess the effect of methylation status on overall survival. We found that 60% (24/40) of breast cancers showed low HOXD13 mRNA expression when compared with corresponding normal breast tissue. The predicted CpG island was located in the -1325 bp to +675 bp region. Next, the -332 bp site in HOXD13 gene promoter was further examined and in 57.7% (113/196) samples methylation was detected at this site. HOXD13 methylation was correlated with larger tumor size (P = 0.004), but not with other clinico-pathological parameters. In addition, patients with methylated -HOXD13 promoter had worse overall survival (OS) (P = 0.005). Based on our results we conclude that HOXD13 methylation is a common event in primary breast cancer and is associated with poor survival of breast cancer patients. HOXD13 methylation could therefore potentially be used as a prognostic factor for breast cancer.     

Semaphorin-3F functions as a tumor suppressor in colorectal cancer due to regulation by DNA methylation

Semaphorin-3F (SEMA3F) is a member of the class III semaphorin family, and is seen as a candidate tumor suppressor gene. The aims of this study were to evaluate the effect of SEMA3F in colorectal cancer (CRC) patients, and to explore the mechanism for that SEMA3F suppresses tumor progression and metastasis. The expression levels of SEMA3F in the colorectal cancer tissues and corresponding non-tumor colorectal tissues were determined by Western blotting and real-time quantitative PCR (qRT-PCR). In addition, we evaluate the effects of SEMA3F on CRC cell migration and colony formation in vitro. Subsequently, quantitative methylation-specific PCR (qMSP) was used to detect the DNA methylation status in the CpG islands of SEMA3F gene promoter in normal colon and colorectal cancer cell lines, colorectal cancer tissues and corresponding non-tumor colorectal tissues. We found that SEMA3F was downregulated in the protein (P < 0.01) and mRNA (P < 0.001) levels in CRC tissues as compared to matched adjacent non-tumor tissues. Moreover, MSP assay showed high levels of SEMA3F gene promoter methylation in the CpG islands in some CRC cell lines and tissue samples. Furthermore, SEMA3F expression was reactivated in CRC cell lines after treatment with 5-Aza-CdR, demethylation of SW620 cells resulted in cell colony formation and invasion inhibition. These findings suggest DNA methylation of promoter CpG island-mediated silencing of the tumor suppressor SEMA3F gene plays an important role in the carcinogenesis of CRC.     

Positive display of methylated sites: a novel method for the detection of promoter methylation.

Promoter methylation represents an important mechanism for silencing gene expression in higher eukaryotes. To study methylation of the promoter of the tumor suppressor p16INK4a, a fast and simple method was developed that, in contrast to previous studies, relies on the positive display of methylated sites (PDM). The method is based on bisulfite treatment of DNA, polymerase chain reaction (PCR)-amplification of the modified DNA and restriction digest of de novo created restriction sites to positively display DNA methylation in a background of unmethylated DNA. Since methylated as well as unmethylated DNA is amplified, information on the proportion of both is provided. Using this approach, 33 ductal invasive carcinomas, 4 normal mammary tissues, and 4 cell lines were analyzed for methylation. Methylation in the p16INK4a promoter was detected in 1 of 33 carcinomas (3%) and in 0 of 4 normal tissues. The conclusion is that PDM provides a useful tool in determining the degree and pattern of promoter methylation and is suitable to screen large series of tissue samples.