Toward a comprehensive and systematic methylome signature in colorectal cancers

CpG Island Methylator Phenotype (CIMP) is one of the underlying mechanisms in colorectal cancer (CRC). This study aimed to define a methylome signature in CRC through a methylation microarray analysis and a compilation of promising CIMP markers from the literature. Illumina HumanMethylation27 (IHM27) array data was generated and analyzed based on statistical differences in methylation data (1st approach) or based on overall differences in methylation percentages using lower 95% CI (2nd approach). Pyrosequencing was performed for the validation of nine genes. A meta-analysis was used to identify CIMP and non-CIMP markers that were hypermethylated in CRC but did not yet make it to the CIMP genes’ list. Our 1st approach for array data analysis demonstrated the limitations in selecting genes for further validation, highlighting the need for the 2nd bioinformatics approach to adequately select genes with differential aberrant methylation. A more comprehensive list, which included non-CIMP genes, such as APC, EVL, CD109, PTEN, TWIST1, DCC, PTPRD, SFRP1, ICAM5, RASSF1A, EYA4, 30ST2, LAMA1, KCNQ5, ADHEF1, and TFPI2, was established. Array data are useful to categorize and cluster colonic lesions based on their global methylation profiles; however, its usefulness in identifying robust methylation markers is limited and rely on the data analysis method. We have identified 16 non-CIMP-panel genes for which we provide rationale for inclusion in a more comprehensive characterization of CIMP+ CRCs. The identification of a definitive list for methylome specific genes in CRC will contribute to better clinical management of CRC patients.     

Identification of novel subregions of LOH in gastric cancer and analysis of the HIC1 and TOB1 tumor suppressor genes in these subregions

Previously, we identified 3 overlapping regions showing loss of heterozygosity (LOH, R(1)-R(3) from 11 to 30 cM) on chromosome 17 in 45 primary gastric cancers (GCs). The data indicated the presence of tumor suppressor genes (TSGs) on chromosome 17 involved in GC. Among the putative TSGs in these regions, HIC1 (in SR(1)) and TOB1 (in SR(3)) remain to be examined in GC. By immunohistochemistry (IHC), methylation-specific PCR (MSP) and western blot, we evaluated the expression and regulation status for HIC1 and TOB1 protein in GC. We narrowed down the deletion intervals on chromosome 17 and defined five smaller LOH subregions, SR(1)-SR(5) (0.54 to 3.42 cM), in GC. We found that HIC1 had downregulated expression in 86% (91/106) and was methylated in 87% (26/30) of primary GCs. Of the primary GCs showing downregulation of HIC1 protein, 75% (18/24) had methylated HIC1 gene. TOB1 was either absent or expressed at reduced levels in 75% (73/97) of the GC samples. In addition, a general reduction was found in total and the ratio of unphosphorylated to phosphorylated TOB1 protein levels in the differentiated GC cell lines. Further analysis revealed significant simultaneous downregulation of both HIC1 and TOB1 protein in GC tissue microarray samples (67%, 52/78) and in primary GCs (65%, 11/17). These results indicate that silencing of HIC1 and TOB1 expression is a common occurrence in GC and may contribute to the development and progression of the disease.     

人参皂苷Rg5对胃癌细胞周期和侵袭的影响及其机制

目的:观察ginsenoside-Rg5 （Rg5）对胃癌细胞周期和侵袭的影响,并探讨其机制。方法:采用不同浓度人参皂苷ginsenoside-Rg3 （Rg3）和Rg5 （10、20、30、40、50μmol/L）处理人正常胃粘膜细胞GES-1和胃癌细胞株AGS、MKN-45 24 h,每个浓度设3个复孔。通过CCK-8检测细胞存活率。通过流式细胞仪检测细胞周期、Transwell小室分析迁移和免疫印迹法及ELISA法检测相关蛋白。结果:CCK8实验结果显示人参皂苷Rg3和Rg5对GES-1细胞无毒副作用,但可以抑制胃癌细胞AGS和MKN-45的增值。且Rg5抗胃癌细胞的活性强于Rg3。20μmol/L Rg5诱导MKN-45细胞发生S期阻滞通过降低Cyclin A1/CDK2/PCNA的表达和升高P21^CIPI蛋白表达。Rg5还可以抑制MKN-45癌细胞的迁移通过降低MMP2和MMP9的表达。WB结果显示Rg5抑制胃癌增殖及迁移主要是通过抑制Notch1蛋白的表达从而调控其下游的周期及侵袭相关蛋白。结论:Rg5抗胃癌细胞活性高于Rg3且通过调控Notch1通路抑制细胞增殖和迁移。     

Patterns of DNA methylation in the normal colon vary by anatomical location,gender, and age

Alterations in DNA methylation have been proposed to create a field cancerization state in the colon, where molecular alterations that predispose cells to transformation occur in histologically normal tissue. However, our understanding of the role of DNA methylation in field cancerization is limited by an incomplete characterization of the methylation state of the normal colon. In order to determine the colon’s normal methylation state, we extracted DNA from normal colon biopsies from the rectum, sigmoid, transverse, and ascending colon and assessed the methylation status of the DNA by pyrosequencing candidate loci as well as with HumanMethylation450 arrays. We found that methylation levels of repetitive elements LINE-1 and SAT-α showed minimal variability throughout the colon in contrast to other loci. Promoter methylation of EVL was highest in the rectum and progressively lower in the proximal segments, whereas ESR1 methylation was higher in older individuals. Genome-wide methylation analysis of normal DNA revealed 8388, 82, and 93 differentially methylated loci that distinguished right from left colon, males from females, and older vs. younger individuals, respectively. Although variability in methylation between biopsies and among different colon segments was minimal for repetitive elements, analyses of specific cancer-related genes as well as a genome-wide methylation analysis demonstrated differential methylation based on colon location, individual age, and gender. These studies advance our knowledge regarding the variation of DNA methylation in the normal colon, a prerequisite for future studies aimed at understanding methylation differences indicative of a colon field effect.     

Patterns of DNA methylation in the normal colon vary by anatomical location,gender, and age

Alterations in DNA methylation have been proposed to create a field cancerization state in the colon, where molecular alterations that predispose cells to transformation occur in histologically normal tissue. However, our understanding of the role of DNA methylation in field cancerization is limited by an incomplete characterization of the methylation state of the normal colon. In order to determine the colon’s normal methylation state, we extracted DNA from normal colon biopsies from the rectum, sigmoid, transverse, and ascending colon and assessed the methylation status of the DNA by pyrosequencing candidate loci as well as with HumanMethylation450 arrays. We found that methylation levels of repetitive elements LINE-1 and SAT-α showed minimal variability throughout the colon in contrast to other loci. Promoter methylation of EVL was highest in the rectum and progressively lower in the proximal segments, whereas ESR1 methylation was higher in older individuals. Genome-wide methylation analysis of normal DNA revealed 8388, 82, and 93 differentially methylated loci that distinguished right from left colon, males from females, and older vs. younger individuals, respectively. Although variability in methylation between biopsies and among different colon segments was minimal for repetitive elements, analyses of specific cancer-related genes as well as a genome-wide methylation analysis demonstrated differential methylation based on colon location, individual age, and gender. These studies advance our knowledge regarding the variation of DNA methylation in the normal colon, a prerequisite for future studies aimed at understanding methylation differences indicative of a colon field effect.     

基于TCGA数据库结肠癌RBL1基因甲基化水平的诊断与预后分析

视网膜母细胞瘤样蛋白1(RBL1/p105)是视网膜母细胞瘤蛋白质家族的成员之一,RBL1通常被认为是抑癌基因,在细胞增殖、凋亡、分化中发挥重要作用。该文探讨RBL1基因在结肠癌诊断和预后中的作用和可能的机制。利用癌症基因组图谱(TCGA)数据库中的结肠癌病例资料和芯片数据,筛选RBL1基因差异甲基化位点,并通过Cox回归模型研究甲基化位点与结肠癌患者预后之间的关系。该研究发现,待研究的癌组织RBL1基因启动子甲基化水平高于癌旁组织(0.120±0.012 vs 0.113±0.008, P=0.000 04)。随后,研究者采用受试者工作特征(ROC)曲线来评价RBL1基因启动子甲基化水平的诊断价值,用曲线下面积(AUC)作为诊断价值的评判标准。研究发现,启动子区甲基化的AUC达到0.732,对应的灵敏度为80.5%、特异度为55.3%。Cox多因素分析结果发现, cg04086771高甲基化是结肠癌患者预后的独立危险因素(P=0.002)。该研究通过对TCGA数据库的挖掘,发现RBL1基因启动子区甲基化均值可能与结肠癌发病风险有关,同时RBL1基因的甲基化位点的甲基化水平对结肠癌的预后有影响。     

胃癌发展相关阶段中E-cadherin基因启动子甲基化及蛋白表达的研究

为了研究E cadherin基因启动子甲基化在胃癌发生及发展阶段中的作用 ,我们采用甲基化特异性PCR和免疫组化的方法对异型增生 (2 3例 )、早期胃癌 (2 0例 )和进展期胃癌 (2 0例 )石蜡标本进行启动子甲基化状态及蛋白表达的分析。结果表明E cadherin基因启动子在异型增生、早期胃癌和进展期胃癌中均有甲基化 ,其阳性率分别为78 3% ,80 %和 90 % ,经χ2 检验各病例组与正常组 (30 % )比较均有差异 (P <0 0 5 ) ,但各病例组间没有差异 (P >0 0 5 ) ;进展期胃癌E cadherin蛋白表达全部阴性 ,早期胃癌 70 %阴性 ,异型增生中无蛋白阴性 ,在早期胃癌和进展期胃癌 34例蛋白表达阴性的标本中 31例有启动子甲基化 (91 2 % ) ,蛋白表达与启动子甲基化呈明显负相关 (P <0 0 1)。表明E cadherin启动子甲基化是胃癌发生的早期事件 ,也是胃癌发生、进展的重要事件     

人类抑癌基因beclin 1在胃癌和直结肠癌中表达下调的研究

人类抑癌基因beclin 1通过自噬作用调节细胞生长,但在胃癌和直结肠癌中其表达水平和调控机制仍不清楚．通过检测胃癌和直结肠肿瘤组织中beclin 1基因的表达水平,及DNA异常甲基化和杂合子缺失对其表达的影响,发现与癌旁组织相比,35％的胃癌标本和30％的直结肠癌标本中beclin 1基因表达显著下调．同时发现,beclin 1基因5’端存在一高密度CpG岛,在胃癌和直结肠癌中beclin 1的启动子区域和第二个内含子区域存在甲基化,而杂合子缺失仅在胃癌中发生．这些发现表明beclin 1基因的异常甲基化和杂合子缺失对其在胃癌和直结肠癌中的表达起调控作用．