乳腺癌中beclin1基因下调机制的研究

目的探讨beclin1在乳腺癌中的可能下调机制。方法用Real-time RT-PCR检测34例乳腺癌中be-clin1 mRNA的表达;Q-PCR分析beclin1是否存在基因的缺失;亚硫酸氢钠测序法检测beclin1基因启动子区域的CpG岛甲基化。结果乳腺癌组织中beclin1的mRNA表达水平与癌旁组织比较显著下调（P=0.005）;Q-PCR发现62%的肿瘤标本中beclin1基因存在缺失;在6例乳腺癌mRNA表达下调的乳腺癌标本中发现启动子区域异常的DNA甲基化。结论beclin1基因的缺失和启动子区域的异常甲基化可能是其在肿瘤细胞中失活的两种机制。     

p16基因甲基化与表达在子宫内膜癌中的意义

肿瘤抑制基因p16定位于9号染色体短臂2区1带,编码细胞周期调节蛋白p16,p16基因失活将导致细胞增殖失控。研究证实肿瘤抑制基因启动子区域5CpG岛甲基化是导致转录水平上基因失活的重要机制。为了研究p16基因甲基化状态及其表达异常与子宫内膜癌发生的关系,采用甲基化特异性PCR(MSP)、免疫组化及PCR方法检测62例子宫内膜癌及相应癌旁组织、10例相应年龄正常子宫内膜中p16基因5′cpG岛甲基化状态、p16蛋白表达及p16基因外显子E,和E：表达缺失情况。结果表明癌旁及正常子宫内膜p16基因无甲基化,且无p16蛋白、外显子1和2的表达异常。62例子宫内膜癌中,15例甲基化,占24、2％;33例p16蛋白表达下降或无表达,占54．8％;p16基因外显子1缺失率16．1％(10／62),外显子2缺失率为30．6％(19／62),两者均缺失9．68％(6／62),至少其中1种缺失46、6％(29／62)。提示P16基因失活在子宫内膜癌中多见且与病理分级、临床分期密切相关。D16基因甲基化在子宫内膜癌的发生中起着重要作用。MSP法测定基因甲基化状态准确且简便可行。     

结肠癌组织RASSF1A基因转录表达和启动子区甲基化研究

目的:研究Ras相关区域家族1A基因(ras association domain family 1A,RASSF1A)启动子区甲基化对结肠癌组织中该基因转录和表达的影响.方法:应用甲基化特异性PCR(Methylation-special PCR,MSP)、RT-PCR和Western blot方法检测30例结肠癌组织和癌旁组织中的RASSF1A基因启动子区甲基化状态、mRNA和蛋白表达水平.结果:①RASSF1A基因启动子区在结肠癌纽织和正常组织中的甲基化频率分别为57%(17/30)和20%(6/30),甲基化频率在两组具有统计学差异(p＜0.01),,结肠癌组织中RASSF1A基因启动子区甲基化频率显著高于癌旁正常组织(x2=8.531,p＜0.01);②结肠癌组织中RASSF1A基因mRNA和蛋白袁达均显著低于癌旁组织(癌组织和癌旁正常组织中mRNA相对表达量分别为0.2836±0.0493和0.5092±0.0433,P＜0.001;以上组织中蛋白相对表达量分别为0.3124±0.0472和0.5320±0.0440,P＜0.01);③在结肠癌组织中,甲基化组RASSF1A基因mRNA和蛋白表达明显低于非甲基化组(甲基化组和非甲基化组mRNA相对表达量分别为0.0686±0.0174和0.5511±0.0486,P＜0.0001;以上组中蛋白相对表达量分别为0.1219±0.0326和0.5614±0.0380,P＜0.0001).结论:结肠癌组织中RASSF1A基因启动子区甲基化明显增高,与该基因蛋白表达减少显著相关,这可能是导致结肠癌中RASSF1A抑癌基因失活的主要因为.     

胃癌组织中MEG3基因甲基化状态及其临床意义

目的：探讨胃癌中MEG3基因差异性甲基化区域甲基化水平及其与MEG3表达之间的关系,并分析其临床病理意义。方法：利用荧光定量PCR检测38例胃癌组织及其对应癌旁正常组织中MEG3的表达水平,并利用甲基化特异性PCR检测MEG3基因差异性甲基化区域的甲基化水平。结果：①胃癌组织中MEG3表达水平明显低于正常癌旁对照组织（P〈0.05）;②胃癌组织MEG3差异性甲基化区域的甲基化率（21/38,55.3%）显著高于正常对照组织（10/38,26.3%,P〈0.05）③胃癌组织MEG3差异性甲基化区域的甲基化率在性别、年龄、发病部位的差异无统计学意义（P〉0.05）;在肿瘤的大小,淋巴结转移,浸润深度上差异有统计学意义（P〈0.05）。结论：胃癌组织中MEG3呈低表达水平,其差异性甲基化区域的甲基化与肿瘤的大小,淋巴结转移,浸润深度有关。     

胃癌组织中MEG3 基因甲基化状态及其临床意义

目的：探讨胃癌中MEG3基因差异性甲基化区域甲基化水平及其与MEG3 表达之间的关系,并分析其临床病理意义。方法： 利用荧光定量PCR 检测38 例胃癌组织及其对应癌旁正常组织中MEG3 的表达水平,并利用甲基化特异性PCR 检测MEG3 基 因差异性甲基化区域的甲基化水平。结果：①胃癌组织中MEG3 表达水平明显低于正常癌旁对照组织（P＜0.05）;②胃癌组织 MEG3差异性甲基化区域的甲基化率（21/38,55.3%）显著高于正常对照组织（10/38,26.3%,P＜0.05）③胃癌组织MEG3差异性甲 基化区域的甲基化率在性别、年龄、发病部位的差异无统计学意义（P＞0.05）;在肿瘤的大小,淋巴结转移,浸润深度上差异有统计 学意义（P＜0.05）。结论：胃癌组织中MEG3 呈低表达水平,其差异性甲基化区域的甲基化与肿瘤的大小,淋巴结转移,浸润深度 有关。     

人胃癌Ha-ras基因及上游区片段甲基化对转化效率及表达的影响

 本文将克隆于pBR322的人胃癌Ha-ras基因(PGC6.6)和带有上游区片段的Ha-ras基因(PGC9.1)的CC~*GG位点甲基化后,转化NIH3T3细胞。发现pGC6.6甲基化与非甲基化对转化效率无明显影响,而pGC9.1甲基化后转化效率明显低于非甲基化pGC9.1者,甲基化/非甲基化pGC9.1的转化效率均明显高于甲基化/非甲基化pGC6.6者。本文又对人胃癌组织及癌旁组织DNA中Ha-ras基因的HpaⅡ、Msp Ⅰ限制性内切酶图谱作了比较,并同对比较了癌及癌旁组织中Ha-ras基因的mRNA水平,发现一例病人癌组织中Ha-ras基因的CC~*GG位点甲基化程度较癌旁组织中者低,且该例中Ha-ras基因表达水平在癌组织中明显地高。这些结果,结合我们以前的研究表明:在人胃Ha-ras癌基因上游区可能存在一增强子样作用的区域,对Ha-ras基因起调控作用。该上游区CC~*GG位点的甲基化能降低这种调控作用。仅Ha-ras结构基因的CC~*GG位点甲基化不足以明显影响其转化活性。在体内,Ha-ras基因甲基化水平降低可能与其达表水平升高以至诱发癌症有关。     

RASSF1A和Runx3基因启动子区甲基化在胃癌组织中的表达及意义

目的：检测胃癌组织中RASSFlA和Runx3基因启动子区甲基化状态,探讨二者与胃癌发生发展的关系。方法：采用甲基化特异性PCR（MSP）技术检测57例胃癌组织和相应癌旁组织及30例正常胃黏膜组织中RASSFlA和Runx3基因启动子区甲基化状态。结果：RASSFlA和Runx3甲基化在正常组未见表达。胃癌组RASSFlA基因甲基化率为64．9％（37／57）,明显高于癌旁组的7．0％（4／57）,差异有统计学意义（P〈0．05）,胃癌组Runx3基因甲基化率为49．1％（28／57）,明显高于癌旁组5．3％（3／57）,差异有统计学意义（P〈O．05）。胃癌组RASSFlA和Runx3基因甲基化率为68．4％（39／57）,明显高于癌旁组的8．8％（5／57）,差异有统计学意义（P〈0．05）。结论：RASSFlA和Runx3基因启动子区高甲基化与胃癌的发生密切相关,有望为胃癌的早期诊治提供理论依据。     

喉鳞癌Apaf-1基因表达及启动子区甲基化研究

喉鳞癌细胞存在多种癌基因和抑癌基因异常,Apaf-1(apoptotic protease activating factor-1)基因是诱导细胞凋亡的肿瘤抑制基因。为探讨Apaf-1基因在喉鳞癌发生中的作用,应用半定量PCR方法分析Apaf-1的表达,用比较基因组杂交(Comparative Genomic Hybrodization,CGH)和杂合性丢失(Loss of Heterozygosity,LOH)分析方法对喉鳞癌病人Apaf-1基因所在的12q22—23区域缺失情况进行研究．并用甲基化特异PCR对该基因启动子区甲基化情况进行了分析。结果表明：11例喉鳞癌组织出现Apaf-1 mRNA表达明显下调,占40．7％(11／27),而6例良性喉肿瘤未发现缺失或下调;CGH分析发现,18例喉鳞癌仅发现2例存在12q22-23区域缺失,未发现扩增;LOH分析发现,72例喉癌组织Apaf-1基因的5个多态位点LOH发生频率低,分别为18．2％(D12S346)、13．9％(D12S1706)、18．2％(D12S327)、22．2％(D12S1657)和16．6％(D12S393),11例出现Apaf-1 mRNA下调的喉癌组织均检测到启动子区甲基化,而16例Apaf-1 mRNA表达未下调者仅1例有甲基化,两者有显著差异(x^2检验,P=0．0001)。通过以上结果,首次证实Apaf-1基因与喉鳞癌相关,在喉鳞癌中Apaf-1基因缺失发生率低．提示启动子区甲基化是该基因失活的首要机制。     

抑癌基因PTEN在胃癌中的研究进展

近年来国内外的学者在多种肿瘤中发现抑癌基因PTEN的等位基因缺失、基因突变、甲基化,PTEN基因杂合性丢失(LOH)频繁的发生于胃癌,而该基因发生突变的频率较低,蛋白表达普遍下降.抑癌基因PTEN异常与胃癌的发生和发展相关,对PTEN功能的进一步研究将为胃癌的诊断和治疗提供新的思路.     

EB病毒阳性和阴性胃癌中视网膜母细胞瘤基因甲基化状态及其表达

目的:了解EB病毒(Epstein-Barr virus,EBV)相关胃癌(EBVaGC)和阴性胃癌(EBVnGC)组织中视网膜母细胞瘤(Rb)基因甲基化状态及蛋白表达,探讨EBV感染与Rb基因甲基化的关系.方法:采用甲基化特异性PCR(MSP)对各种临床病理指标匹配的23例EBVaGC和25例EBVnGC组织及相应癌旁组织中Rb基因启动子区域的甲基化状态进行检测,并采用免疫组化技术检测两种胃癌组织中Rb蛋白的表达.结果:胃癌与相应癌旁组织中Rb基因启动子区的甲基化率分别为64.6%(31/48)和39.6%(19/48),差异有显著性(P<0.05);EBVaGC组织中Rb基因启动子区的甲基化率为82.6%(19/23),高于EBVnGC中的检出率(48.0%,12/25),差异有显著性(P<0.05);EBVaGC和EBVnGC组织中Rb蛋白的表达率分别为52.2%(12/23)和72.0%(18/25),差异无显著性(P□0.05);胃癌组织中Rb启动子基因甲基化与蛋白表达无明显负相关.结论:Rb异常甲基化在胃癌细胞中较常见,EBV可以诱导Rb基因甲基化,影响其基因表达而参与EBVaGC的发生.     

Association of NDRG1 Gene Promoter Methylation with Reduced NDRG1 Expression in Gastric Cancer Cells and Tissue Specimens

NDRG1 (N-myc downstream-regulated gene 1) plays a role in cell differentiation and suppression of tumor metastasis. This study aims to determine the expression of NDRG1 mRNA and protein in gastric cancer cell lines and tissue specimens and then assess the possible cause of its aberrant expression. Six gastric cancer cell lines and 20 pairs of normal and gastric cancer tissue samples were used to assess NDRG1 expression using Real-time PCR and Western blot. High-resolution melting analysis (HRM) and methylation-specific PCR (MSP) were performed to detect gene mutation and methylation, respectively, in cell lines and tissues samples. Expression of NDRG1 mRNA and protein was downregulated in gastric cancer cell lines and tissues. Specifically, expression of NDRG1 mRNA and protein was lower in all six gastric cancer cell lines than that of normal gastric cells, while 15 out of 20 cases of gastric cancer tissues had the reduced levels of NDRG1 mRNA and protein. HRM data showed that there was no mutation in NDRG1 gene, but MSP data showed high levels of NDRG1 gene promoter methylation in the CpG islands in both cell lines and tissue samples. Moreover, treatment with the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine upregulated NDRG1 expression in gastric cancer HGC27 cells, but not in the histone deacetylase inhibitor trichostatin A-treated HGC27 cells. In conclusion, this study has shown that expression of NDRG1 mRNA and protein was reduced in gastric cancer cell lines and tissues, which is due to methylation of NDRG1 gene promoter. Further study will unearth the clinical significance of the reduced NDRG1 protein in gastric cancer.     

Epigenetic silencing of Notch signaling in gastrointestinal cancers

The Notch signaling pathway drives proliferation, differentiation, apoptosis, cell fate choices and maintenance of stem cells during embryogenesis and in self-renewing tissues of the adult. In addition, aberrant Notch signaling has been implicated in several tumors, where Notch can function both as an oncogene or a tumor-suppressor gene, depending on the context. This Extra View aims to review what is currently known about Notch signaling, in particular in gastrointestinal tumors, providing a summary of our data on Notch1 signaling in gastric cancer with results obtained in colorectal cancer (CRC). We have already reported that the epigenetic regulation of the Notch ligand DLL1 controls Notch1 signaling activation in gastric cancer, and that Notch1 inhibition is associated with the diffuse type of gastric cancer. Here, we describe additional data showing that in CRC cell lines, unlike gastric cancer, DLL1 expression is not regulated by promoter methylation. Moreover, in CRC, Notch1 receptor is not affected by any mutation. These data suggest a different regulation of Notch1 signaling between gastric cancer and CRC.     

Frequent Epigenetic Silencing of the Folate-Metabolising Gene Cystathionine-Beta-Synthase in Gastrointestinal Cancer

Background

Both gastric and colorectal cancers (CRC) are the most frequently occurring malignancies worldwide with the overall survival of these patients remains unsatisfied. Identification of tumor suppressor genes (TSG) silenced by promoter CpG methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic biomarkers for early cancer detection and prognosis assessment. Cystathionine-beta-synthase (CBS) functions in the folate metabolism pathway, which is intricately linked to methylation of genomic DNA. Dysregulation of DNA methylation contributes substantially to cancer development.

Methodology/Principal Findings

To identify potential TSGs silenced by aberrant promoter methylation in CRC, we analyzed tumor and adjacent tissues from CRC cases using the Illumina Human Methylation45 BeadChip. We identified hypermethylation of the CBS gene in CRC samples, compared to adjacent tissues. Methylation and decreased mRNA expression of CBS were detected in most CRC cell lines by methylation-specific PCR and semiquantitative RT-PCR, as well as in gastric cancer. Treatment with 5-aza-2''-deoxycytidine and/or trichostatin A reversed methylation and restored CBS mRNA expression indicating a direct effect. Aberrant methylation was further detected in 31% of primary CRCs (29 of 96) and 55% of gastric tumors (11 of 20). In contrast, methylation was seldom found in normal tissues adjacent to the tumor. CBS methylation was associated with KRAS mutations in primary CRCs (P = 0.04, by χ²-test). However, no association was found between CBS methylation or KRAS mutations with cancer relapse/metastasis in Stage II CRC patients.

Conclusion

A novel finding from this study is that the folate metabolism enzyme CBS mRNA levels are frequently downregulated through CpG methylation of the CBS gene in gastric cancer and CRC, suggesting that CBS functions as a tumor suppressor gene. These findings warrant further study of CBS as an epigenetic biomarker for molecular diagnosis of gastrointestinal cancers.     

Deregulated Expression of SRC,LYN and CKB Kinases by DNA Methylation and Its Potential Role in Gastric Cancer Invasiveness and Metastasis

Kinases are downstream modulators and effectors of several cellular signaling cascades and play key roles in the development of neoplastic disease. In this study, we aimed to evaluate SRC, LYN and CKB protein and mRNA expression, as well as their promoter methylation, in gastric cancer. We found elevated expression of SRC and LYN kinase mRNA and protein but decreased levels of CKB kinase, alterations that may have a role in the invasiveness and metastasis of gastric tumors. Expression of the three studied kinases was also associated with MYC oncogene expression, a possible biomarker for gastric cancer. To understand the mechanisms that regulate the expression of these genes, we evaluated the DNA promoter methylation of the three kinases. We found that reduced SRC and LYN methylation and increased CKB methylation was associated with gastric cancer. The reduced SRC and LYN methylation was associated with increased levels of mRNA and protein expression, suggesting that DNA methylation is involved in regulating the expression of these kinases. Conversely, reduced CKB methylation was observed in samples with reduced mRNA and protein expression, suggesting CKB expression was found to be only partly regulated by DNA methylation. Additionally, we found that alterations in the DNA methylation pattern of the three studied kinases were also associated with the gastric cancer onset, advanced gastric cancer, deeper tumor invasion and the presence of metastasis. Therefore, SRC, LYN and CKB expression or DNA methylation could be useful markers for predicting tumor progression and targeting in anti-cancer strategies.     

Epigenetic silencing of Notch signaling in gastrointestinal cancers

The Notch signaling pathway drives proliferation, differentiation, apoptosis, cell fate choices and maintenance of stem cells during embryogenesis and in self-renewing tissues of the adult. In addition, aberrant Notch signaling has been implicated in several tumors, where Notch can function both as an oncogene or a tumor-suppressor gene, depending on the context.

This Extra View aims to review what is currently known about Notch signaling, in particular in gastrointestinal tumors, providing a summary of our data on Notch1 signaling in gastric cancer with results obtained in colorectal cancer (CRC).

We have already reported that the epigenetic regulation of the Notch ligand DLL1 controls Notch1 signaling activation in gastric cancer, and that Notch1 inhibition is associated with the diffuse type of gastric cancer. Here, we describe additional data showing that in CRC cell lines, unlike gastric cancer, DLL1 expression is not regulated by promoter methylation. Moreover, in CRC, Notch1 receptor is not affected by any mutation. These data suggest a different regulation of Notch1 signaling between gastric cancer and CRC.     

Frequent promoter hypermethylation and expression reduction of the glucocorticoid receptor gene in breast tumors

Previous studies have found that expression of the Glucocorticoid Receptor (GR) is altered or reduced in various cancers, while the GR promoter has been shown to be methylated in gastric, lung, and colorectal cancers. Examining a small cohort of matched normal and breast cancer samples we found that GR levels were dramatically reduced in almost all tumors in relation to their normal tissue. The methylation status of the GR promoter was assessed to determine if this observed decrease of expression in breast tumors could be due to epigenetic regulation. While it was not methylated in normal tissue, the GR proximal promoter was methylated in 15% of tumor samples, particularly, but not exclusively, in Estrogen Receptor positive tumors. GR expression in these tumors was particularly low and loss of GR expression was specifically correlated with methylation of the proximal promoter GR B region. Overall, these results show that hypermethylation of the promoter in tumors is a frequent event and suggests that GR may act as a tumor suppressor in breast tissue.     

Frequent promoter hypermethylation and expression reduction of the glucocorticoid receptor gene in breast tumors

Previous studies have found that expression of the Glucocorticoid Receptor (GR) is altered or reduced in various cancers, while the GR promoter has been shown to be methylated in gastric, lung, and colorectal cancers. Examining a small cohort of matched normal and breast cancer samples we found that GR levels were dramatically reduced in almost all tumors in relation to their normal tissue. The methylation status of the GR promoter was assessed to determine if this observed decrease of expression in breast tumors could be due to epigenetic regulation. While it was not methylated in normal tissue, the GR proximal promoter was methylated in 15% of tumor samples, particularly, but not exclusively, in Estrogen Receptor positive tumors. GR expression in these tumors was particularly low and loss of GR expression was specifically correlated with methylation of the proximal promoter GR B region. Overall, these results show that hypermethylation of the promoter in tumors is a frequent event and suggests that GR may act as a tumor suppressor in breast tissue.     

Down-regulation of thyroid hormone receptor β1 gene expression in gastric cancer involves promoter methylation

Hypermethylation has been shown in the promoter region of the thyroid hormone receptor β1 (TRβ1) gene in several human tumors. However, its role in gastric cancer formation is still unclear. In the study, we analyzed mRNA expression of TRβ1 gene using real-time quantitative PCR (qPCR). A quantitative methylation-specific PCR (Q-MSP) assay was used to determine the methylation status of the TRβ1 gene promoter region in 46 pair-matched gastric neoplastic and adjacent non-neoplastic tissues. The results showed that TRβ1 mRNA expression was significantly reduced in gastric cancer specimens. The methylation of promoter of TRβ1 gene in gastric cancer tissues was significantly higher than in adjacent normal tissues. Promoter hypermethylation of the TRβ1 gene correlated with tumor infiltration, lymph node metastasis, and distant metastasis, but it was not associated with other clinicopathological characteristics. We treated gastric cancer cell lines MKN-45, MKN-28, SGC-7901, NCI-N87, and SNU-1 with 5-Aza-2-deoxycytidine (5-Aza-dC). The results showed the expression of TRβ1 mRNA was increased in MKN-45, MKN-28, SGC-7901, but not increased in NCI-N87 and SNU-1. These results suggest that the TRβ1 gene plays important roles in the development of gastric cancer partially through epigenetic mechanisms.