Quantitative assessment of gene methylation in neoplastic and non‐neoplastic gastric epithelia using methylation‐specific DNA microarray

A fiber‐type DNA microarray was used to calculate methylation rates (MR) of four tumor suppressor genes, lysyl oxidase (LOX), p16, RUNX3, and tazarotene‐induced gene 1 (TIG1). MR were calculated in 26 primary gastric cancers and corresponding non‐neoplastic gastric epithelia, and the results were compared to those of conventional methylation‐specific polymerase chain reaction (MSP). MR ranged from 0.1% to 69.1% (mean, 18.3%) for LOX, 0.5–74.1% (mean, 15.7%) for p16, 0.2–76.5% (mean, 22.7%) for RUNX3, and 0.6–41.2% (mean, 5.8%) for TIG1 in primary gastric cancers, and from 0.1% to 25.8% (mean, 8.7%) for LOX, 1.0– 23.2% (mean, 10.3%) for p16, 0.7–25.1% (mean, 5.5%) for RUNX3, and 1.8–27.6% (mean, 11.4%) for TIG1 in corresponding non‐neoplastic gastric epithelia. Although MR varied significantly across different samples for both neoplastic and non‐neoplastic gastric epithelia, high‐level methylation (MR >40%) was cancer specific and was observed in 19.2%, 19.2%, 30.8%, and 3.8% of primary gastric cancers for LOX, p16, RUNX3, and TIG1, respectively. All samples with high‐level methylation, as well as some samples with low MR (particularly <10%) were judged to be methylation positive on conventional MSP. Quantitative analysis of gene methylation using methylation‐specific DNA microarray is a promising method for cancer diagnosis.     

RUNX3基因启动子甲基化与胃癌关系的meta分析

目的采用meta分析的方法评价RUNX3基因启动子甲基化与胃癌发生的关系。方法检索公开发表在Pubmed、EMBASE、Ovid、中国期刊全文数据库（CNKI）关于RUNX3基因启动子甲基化与胃癌发生关系的研究。应用Statall．0统计软件分析RUNX3基因启动子甲基化与胃癌发生间的关联。结果共有19项研究纳入分析．meta分析结果显示：胃癌患者癌组织中RUNX3基因启动子甲基化发生率为P=55％（95％CI：45％～66％）：胃癌患者远癌正常胃组织中RUNX3基因启动子甲基化发生率为P=18％（95％CI：7％～29％）。与远癌正常胃组织相比,胃癌组织中RUNX3基因启动子甲基化发生优势比OR=7．85（95％CI：5．81～10．61）。结论胃癌患者癌组织中RUNX3基因启动子甲基化发生率明显高于远癌正常胃组织,RUNX3基因启动子甲基化可能与胃癌的发生密切相关。     

RUNX3基因与胃癌的关系

RUNX3基因是RUNX转录因子家族成员之一，其在胃黏膜上皮生长调控、脊神经节的神经发育和T细胞分化过程中发挥重要作用。在人类多种恶性肿瘤尤其是胃癌中发现RUNX3基因表达缺失或下调。目前发现有多种机制包括杂合性缺失、高甲基化和点突变等参与了RUNX3基因在胃癌中的表达缺失或下调，其中RUNX3启动子区域CpG岛的甲基化是导致其在胃癌中失活的主要机制。随着研究的不断深入，RUNX3基因有望成为胃癌诊断的一个特异性生物学标志物和基因治疗的靶点。     

Prediction of the risk for gastric cancer using candidate methylation markers in the non-neoplastic gastric mucosae

Aberrant DNA methylation is frequently found during gastric carcinogenesis. Recently, we identified potential methylation markers important for Helicobacter pylori-induced gastric carcinogenesis using an Illumina methylation chip assay. In this study, we evaluated the candidate genes as markers for gastric cancer (GC) in a large Korean population. DNA methylation of PTPN6, MOS, DCC, CRK, and VAV1 was evaluated in non-neoplastic gastric specimens using quantitative methylation-specific PCR in patients with GC (n = 207) and their age- and gender-matched controls (n = 207). Methylation levels in 125 GC samples were also compared. H. pylori infection status was categorized as negative, active, or past infection according to the results of endoscopy-based tests (CLOtest, histology, and culture), H. pylori serology, and serum pepsinogen test. In the controls, active H. pylori infection increased methylation levels in DCC, CRK, MOS, and VAV1 but decreased methylation levels in PTPN6 (all p < 0.05); the methylation levels in MOS remained increased in patients with past H. pylori infection compared to H. pylori-negative subjects (p < 0.001). Methylation levels in MOS in non-neoplastic gastric mucosae increased in the presence of GC, regardless of H. pylori infection status (p < 0.01). Methylation levels in all genes but DCC decreased significantly in GC specimens compared to neoplastic gastric mucosae (p < 0.01); however, methylation levels in GC tissues were not correlated with those in their background gastric mucosae. Hypomethylation of MOS in GC tissues was associated with tumour invasion, nodal metastasis, and undifferentiated histology (p < 0.05). To summarize, among the candidate genes, DNA methylation of MOS may reflect the duration of H. pylori exposure and may be a marker for the development of GC.     

胃癌组织中RUNX3与mP53表达的关系及意义

目的观察RUNX3蛋白在正常胃黏膜、癌前病变及胃癌组织中的表达,分析RUNX3与mP53在胃癌中表达的关系,探讨Runx3与胃癌临床病理因素的关系以及二者在胃癌发生发展中的作用和意义。方法采用免疫组化PV9000法检测91例胃癌及配对正常胃黏膜,19例肠上皮化生,5例异型增生中RUNX3和mP53的表达。结果RUNX3在胃癌组织中的阳性表达率(48/91,52.75%,P<0.05)显著低于正常胃黏膜(86/91,94.51%)和肠上皮化生(19/19,100%)。RUNX3与胃癌临床病理因素未见显著相关性。胃癌组织中mP53的阳性表达率随分化程度降低而升高,但无统计学意义。胃癌组织中RUNX3与mP53表达呈负相关(=-0.225,P<0.05)。结论胃癌组织中RUNX3表达缺失与mP53过表达可能参与胃黏膜的癌变和胃癌的发病机制和演进。     

DNA甲基化和组蛋白乙酰化与胃癌的研究进展

胃癌是我国最常见的恶性肿瘤，但其具体发病机制还不是很清楚。表遗传学改变，如DNA甲基化和组蛋白修饰改变可以调节基因的表达，在肿瘤的发生和发展中可能起关键作用。近年来一些胃癌相关基因的DNA甲基化和组蛋白乙酰化改变已经得到证实，这些基因改变涉及细胞凋亡、细胞周期阻滞、分化和增殖等。     

RUNX3基因表达对判断人乳腺癌预后的价值

目的探讨乳腺癌组织中RUNX3基因的表达及其与乳腺癌生物学特征和预后的关系。方法采用免疫组化SP法检测RUNX3蛋白在88例乳腺癌、40例乳腺纤维腺瘤和40例乳腺增生病组织中的表达。结果（1）RUNX3在乳腺癌中的阳性表达率为35．23％,明显低于在乳腺纤维腺瘤（85％）及乳腺增生病（87．5％）组织中的表达率,差异具有统计学意义（P〈0．05）。（2）RUNX3蛋白表达与乳腺癌有无浸润、临床分期、淋巴结转移、ER、PR表达相关,而与病人的年龄、肿瘤类型、病理分级无关。（3）RUNX3表达阳性者的：生存率高于表达阴性者的生存率（P〈0．05）。RUNX3阳性表达者,术后生存时间长。结论（1）乳腺癌组织中RUNX3蛋白表达降低,证明RUNX3基因可能作为一个抑癌基因参与乳腺癌的发生。（2）随着乳腺癌的临床进展,RUNX3表达下降。（3）RUNX3在乳腺癌中的表达对评价患者的预后有一定价值。     

DNA methyltransferase 3a rs1550117 genetic polymorphism predicts poor survival in gastric cancer patients

DNA methyltransferase 3a (DNMT3a) have been suggested to play a crucial role in human cancer prognosis. Single nucleotide polymorphisms (SNPs) in DNMT3a genes may have an impact on the prognosis of cancers. This study aimed to investigate the association between SNPs of DNMT3a gene and prognosis of gastric cancer (GC). Two sites of DNMT3a SNPs, rs1550117 and rs13420827 were selected and genotyped using TaqMan assay in 447 GC patients who received gastrectomy. Effects of genotypes on clinical outcomes of GC were calculated by Kaplan-Meier survival analysis and Cox regression model. We found that the AG or AA genotype of rs1550117 was associated with significantly poorer survival and increased death risk of GC compared with GG genotype (dominant model: HR=1.35, 95% CI=1.01-1.80, P=0.043). Further multivariate Cox regression analysis revealed that in addition to the known factors including male, larger tumor sizes and high clinical stage, rs1550117 variant was an independently predictive factor for survival in GC patients. No significant association was found between rs13420827 genetic variants and GC prognosis. Our findings first demonstrated that DNMT3a rs1550117 polymorphism may be a potential biomarker in predicting overall survival of GC patients.     

Epstein–Barr virus infection induces genome‐wide de novo DNA methylation in non‐neoplastic gastric epithelial cells

Epstein–Barr virus (EBV)‐positive gastric cancer (GC) shows a higher DNA methylation epigenotype. EBV infection can causally induce genome‐wide aberrant DNA methylation, as previously demonstrated by in vitro infection experiments in the low‐methylation GC cell line MKN7. However, whether EBV exerts DNA methylation remodelling properties in non‐neoplastic epithelial cells remains unclear. Here we performed post‐infection time‐series DNA methylation analyses using the immortalized normal gastric epithelial cell line GES1. Genome‐wide analysis using Illumina's Infinium 450 k BeadArray demonstrated global de novo DNA methylation from post‐infection day 17, which was completed by 28 days in a manner similar to that observed in MKN7 cells. De novo methylation of all types of GC‐specific methylation marker genes was observed, indicating that EBV infection is sufficient for gastric epithelial cells to acquire an EBV‐positive GC epigenotype. Pyrosequencing demonstrated that methylation of the viral genome preceded that of the host cellular genome, suggesting the existence of well‐ordered mechanisms that induce methylation. Spatiotemporal representation with differential models revealed dynamic alterations of DNA methylation in promoter regions, occurring from lower‐CpG peripheral regions and extending to higher‐CpG core regions. In summary, EBV infection exerted powerful pressure to induce global de novo DNA methylation in non‐neoplastic cells within a month in a spatiotemporally well‐ordered manner. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

胃癌相关抑癌基因甲基化研究进展

DNA甲基化是一种表观遗传修饰,它与肿瘤的发生关系密切。DNA甲基化是基因表达调控的一种方式。抑癌基因启动子高甲基化可以使其表达失活,导致肿瘤发生。胃癌的发生与多基因异常表达密切相关,其中抑癌基因甲基化是胃癌发生发展的重要机制之一。     

DNA微阵列技术在胃癌研究中的应用

多年来作为全球第二大恶性肿瘤,胃癌的研究急需一种更为整体性的研究方法。DNA微阵列技术以其高通量、自动化、平行化、快速化的优势,成为后基因组时代在转录组水平进行肿瘤基因表达谱分析的最强有力工具。本文介绍了DNA微阵列技术的应用原理和实验方法,着重回顾了这项技术在胃癌研究中的应用进展情况,并对现存的问题和应用前景进行了评价。     

单增李斯特菌检测基因芯片的研制与评价

目的 研制快速、特异、灵敏的检测单增李斯特菌的基因芯片.方法 选择gyrB、ISR、16S rRNA、23S rRNA、hlyA、iap和prfA作为单增李斯特菌的检测靶基因,研制一种Oligo探针基因芯片,对18个不同种属来源的已知参考生物样品进行检测和鉴定,并且采用对比试验、重复性试验、灵敏度试验和特异性试验对该芯片进行验证评估.结果 通过对比发现IDT合成的70 mer Oligo芯片探针在芯片打印与芯片检测两个方面较优.比较10、40和80 μmol/L 3个Oligo探针点样浓度,结果 显示10 μmol/L的探针点样浓度已能获得很好的芯片检测结果.单增李斯特菌检测芯片具有较好的重复性;样品检测绝对量下限为0.9 ng DNA左右.结论 Oligo基因芯片可以快速准确地检测单增李斯特菌.     

白血病Reh、HL-60、K562细胞系RUNX 3 基因P2启动子甲基化状态与RUNX 3 基因表达

目的: 研究人白血病Reh、HL-60与K562细胞系RUNX 3 基因P2启动子甲基化状态及RUNX 3 基因的表达,分析P2启动子甲基化与基因表达之间的关系。方法: 运用甲基化特异性PCR(MSP)和RT-PCR检测Reh、HL-60与K562细胞系RUNX 3 基因P2启动子的甲基化状态及RUNX 3 基因的表达。结果: Reh及K562细胞系RUNX 3 基因P2启动子甲基化特异性扩增呈阳性,非甲基化特异性扩增呈阴性,RT-PCR扩增呈阴性;而HL-60细胞系相反,甲基化特异性扩增呈阴性,而非甲基化特异性扩增呈阳性,RT-PCR扩增阳性。结论: Reh及K562细胞中存在RUNX 3 基因P2启动子的甲基化,且在不同类型白血病细胞系中的甲基化状态不同。     

胃癌中RUNX3、cyclin E和P21蛋白表达与患者生存的关系

目的：探讨胃癌RUNX3蛋白表达对细胞周期蛋白的影响及其与胃癌患者生存和生物学特性的关系。方法:应用免疫组化，分析RUNX3蛋白的表达；应用流式细胞术，分析cyclinE和P21蛋白表达；采用Kaplan-Meier限乘法计算生存率。结果:在56例胃癌中RUNX3蛋白、cyclinE和P21蛋白的阳性表达率分别为44.6%、64.3%和32.1%。胃癌细胞RUNX3蛋白表达与淋巴转移和远处转移有关(P<0.05)。胃癌细胞cyclinE的表达与浸润深度、淋巴转移和远处转移有关(P<0.05)。而胃癌细胞P21的表达与远处转移有关(P<0.05)。胃癌细胞中RUNX3和P21的表达之间呈明显正相关(r=0.57,P<0.05)，而与 cyclinE的表达之间无相关性(r=0.25,P>0.05)。用Kaplan-Meier生存曲线经Log-rank检验发现，RUNX3和cyclinE的阳性表达与生存相关(P<0.05)，P21的阳性表达与生存无关(P>0.05) 。结论：RUNX3蛋白可能通过影响P21蛋白的表达影响胃癌的发生发展；检测RUNX3和cyclinE的表达可作为反映胃癌临床病理学特点和预后的指标。     

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.     

Lack of RUNX3 inactivation in columnar cell lesions of breast

Subramaniam M M, Chan J Y, Omar M F M, Ito K, Ito Y, Yeoh K G, Salto‐Tellez M & Putti T C
(2010) Histopathology 57 , 555–563
Lack of RUNX3 inactivation in columnar cell lesions of breast Aims: Ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) exhibit frequent RUNX3 inactivation by promoter hypermethylation and protein mislocalization. The aim of this study was to analyse columnar cell lesions (CCLs) to further characterize RUNX3 involvement in breast carcinogenesis. Methods and results: RUNX3 expression and methylation was analysed by immunohistochemistry and methylation‐specific polymerase chain reaction (PCR), respectively, in 75 CCLs. Our previously reported DCIS and IDC data were also included. Consistent with terminal duct lobular units (TDLUs) (73 of 75, 97%), active nuclear RUNX3 protein was observed in 73 of 75 (97%) CCLs [columnar cell change, 46 of 48 (96%); columnar cell hyperplasia, 12 of 12 (100%) and flat epithelial atypia, 15 of 15 (100%). In contrast to matched TDLUs from cancer specimens [four of 40 (10%)] and CCLs, significantly inactivated RUNX3 expression was detected in DCIS [17 of 20 (85%)] and IDC [18 of 20 (90%)] (all P < 0.001). RUNX3 methylation was more frequent in DCIS [15 of 20 (75%)] and IDC [16 of 20 (80%)] than CCLs [(none of 20 (0%)] and matched TDLUs [one of 10 (10%)] from cancer patients (all P < 0.001). Conclusions: RUNX3 inactivation occurs specifically in DCIS and IDC cells. In addition, RUNX3 inactivation may not be a common association between CCLs and breast carcinomas.