DNA低甲基化与胃癌发病的关系

随着对胃癌研究的不断深人，人们逐渐开始关注DNA甲基化这种表观遗传学改变在胃癌发病中所起到的作用。DNA甲基化异常有高甲基化和低甲基化之分，两者均与胃癌的发病密切相关。其中DNA低甲基化主要通过影响胃癌原癌基因、肿瘤-睾丸抗原及浸润、转移相关基因的表达，在胃癌的发生发展中发挥重要作用。     

Role of site-specific promoter hypomethylation in aberrant MUC2 mucin expression in mucinous gastric carcinomas

In the present work we investigated the methylation levels of mucin gene (MUC)2 promoter region in normal gastric mucosa and mucinous gastric carcinoma in order to access if the observed de novo expression of the intestinal mucin MUC2 in mucinous gastric carcinoma is associated with changes in MUC2 promoter methylation status. The results obtained by methylation-sensitive single nucleotide primer extension suggest that MUC2 expression in gastric cells is regulated by promoter methylation and further indicate that two specific cytosine guaine dinucleotide (CpG) sites may play a particularly important regulatory role.     

Gene Expression Profile Differences in Gastric Cancer and Normal Gastric Mucosa by Oligonucleotide Microarrays

OBJECTIVE To study the difference of gene expression in gastric cancer (T) and normal tissue of gastric mucosa (C), and to screen for associated novel genes in gastric cancers by oligonucleotide microarrays. METHODS U133A (Affymetrix, Santa Clara, CA) gene chip was used to detect the gene expression profile difference in T and C. Bioinformatics was used to analyze the detected results. RESULTS When gastric cancers were compared with normal gastric mucosa, a total of 270 genes were found with a difference of more than 9 times in expression levels. Of the 270 genes, 157 were up-regulated (Signal Log Ratio [SLR] ≥3), and 113 were down-regulated (SLR ≤-3). Using a classification of function, the highest number of gene expression differences related to enzymes and their regulatory genes (67, 24.8%), followed by signal-transduction genes (43,15.9%). The third were nucleic acid binding genes (17, 6.3%), fourth were transporter genes (15, 5.5%) and fifth were protein binding genes (12, 4.4%). In addition there were 50 genes of unknown function, accounting for 18.5%. The five above mentioned groups made up 56.9% of the total gene number. CONCLUSION The 5 gene groups (enzymes and their regulatory proteins, signal transduction proteins, nucleic acid binding proteins, transporter and protein binding) were abnormally expressed and are important genes for further study in gastric cancers.     

Analysis of Metastatic-Related Gene Expression in Gastric Cancer by Low-Density cDNA Microarrays

OBJECTIVE To screen metastatic-related genes in human gastric cancer by a low-density cDNA microarray technique. METHODS A total of 18 paired gastric cancer and adjacent normal mu-cosa were examined by a low-density cDNA microarray containing 23 genes. RT-PCR was used for further verification. RESULTS The mRNA expression of MMP -7, heparanase, S100A4, hTERT, hRad17 in gastric cancers was higher than that in coupled normal mucosa (P=0.002, 0.00011, 0.000072, 0.002, 0.00016 respectively), whereas nm23H1, and CDH1 were lower (P=0.003, 0.012 respectively). The concordance was verified further by RT-PCR with a correlation coefficient of 0.774. In gastric primary lesions the mRNA expression of MMP-7, heparanase and S100A4 was higher in the serosa involved compared to non-involved (P=0.003, 0.009, 0.012 respectively), whereas nm23H1, CDH1, KAI1 were lower (P=0.001, 0.001, 0.006 respectively). With respect to the area of serosa involvement, MMP-7 and heparanase expressions were higher in an area of more than 20 cm2 compared to an area of less than 20 cm2 (P=0.001, 0.02 respectively), whereas nm23H1, CDH1 and KAI1 were lower (P=0.030, 0.041, 0.031 respectively). MMP-7 and hTERT expressions were higher in the heavier lymph node metastatic cases (no less than 7) than in the lighter lymph node metastatic cases (no more than 6, P=0.001, 0.005 respectively). CONCLUSION Expression of MMP -7, S100A4, heparanase, hTERT, KAI1, CDH1 and nm23H1 correlated closely with invasion and metastasis in gastric carcinomas. The low-density cDNA microarrays can be used to examine the expression of many genes simultaneously, parallely and quickly.     

Global gene expression analysis of gastric cancer by oligonucleotide microarrays.

To gain molecular understanding of carcinogenesis, progression, and diversity of gastric cancer, 22 primary human advanced gastric cancer tissues and 8 noncancerous gastric tissues were analyzed by high-density oligonucleotide microarray in this study. Based on expression analysis of approximately 6800 genes, a two-way clustering algorithm successfully distinguished cancer tissues from noncancerous tissues. Subsequently, genes that were differentially expressed in cancer and noncancerous tissues were identified; 162 and 129 genes were highly expressed (P < 0.05) >2.5-fold in cancer tissues and noncancerous tissues, respectively. In cancer tissues, genes related to cell cycle, growth factor, cell motility, cell adhesion, and matrix remodeling were highly expressed. In noncancerous tissues, genes related to gastrointestinal-specific function and immune response were highly expressed. Furthermore, we identified several genes associated with lymph node metastasis including Oct-2 or histological types including Liver-Intestine Cadherin. These results provide not only a new molecular basis for understanding biological properties of gastric cancer, but also useful resources for future development of therapeutic targets and diagnostic markers for gastric cancer.     

DNA hypomethylation and ovarian cancer biology

Hypomethylation of some portions of the genome and hypermethylation of others are very frequent in human cancer. The hypomethylation often involves satellite 2 (Sat2) DNA in the juxtacentromeric (centromere-adjacent) region of chromosome 1. In this study, we analyzed methylation in centromeric and juxtacentromeric satellite DNA in 115 ovarian cancers, 26 non-neoplastic ovarian specimens, and various normal somatic tissue standards. We found that hypomethylation of both types of satellite DNA in ovarian samples increased significantly from non-neoplastic toward cancer tissue. Furthermore, strong hypomethylation was significantly more prevalent in tumors of advanced stage or high grade. Importantly, extensive hypomethylation of Sat2 DNA in chromosome 1 was a highly significant marker of poor prognosis (relative risk for relapse, 4.1, and death, 9.4) and more informative than tumor grade or stage. Also, comparing methylation of satellite DNA and 15 5' gene regions, which are often hypermethylated in cancer or implicated in ovarian carcinogenesis, we generally found no positive or negative association between methylation changes in satellite DNA and in the gene regions. However, hypermethylation at two loci, CDH13 (at 16q24) and RNR1 (at 13p12), was correlated strongly with lower levels of Sat2 hypomethylation. The CDH13/Sat2 epigenetic correlation was seen also in breast cancers. We conclude that satellite DNA hypomethylation is an important issue in ovarian carcinogenesis as demonstrated by: (a) an increase from non-neoplastic tissue toward ovarian cancer; (b) an increase within the ovarian cancer group toward advanced grade and stage; and (c) the finding that strong hypomethylation was an independent marker of poor prognosis.     

Gene expression profiling of colon cancer by DNA microarrays and correlation with histoclinical parameters

Different diagnostic and prognostic groups of colorectal carcinoma (CRC) have been defined. However, accurate diagnosis and prediction of survival are sometimes difficult. Gene expression profiling might improve these classifications and bring new insights into underlying molecular mechanisms. We profiled 50 cancerous and noncancerous colon tissues using DNA microarrrays consisting of approximately 8000 spotted human cDNA. Global hierarchical clustering was to some extent able to distinguish clinically relevant subgroups, normal versus cancer tissues and metastatic versus nonmetastatic tumours. Supervised analyses improved these segregations by identifying sets of genes that discriminated between normal and tumour tissues, tumours associated or not with lymph node invasion or genetic instability, and tumours from the right or left colon. A similar approach identified a gene set that divided patients with significantly different 5-year survival (100% in one group and 40% in the other group; P=0.005). Discriminator genes were associated with various cellular processes. An immunohistochemical study on 382 tumour and normal samples deposited onto a tissue microarray subsequently validated the upregulation of NM23 in CRC and a downregulation in poor prognosis tumours. These results suggest that microarrays may provide means to improve the classification of CRC, provide new potential targets against carcinogenesis and new diagnostic and/or prognostic markers and therapeutic targets.     

Insulin-like growth factor 2 hypomethylation of blood leukocyte DNA is associated with gastric cancer risk

To determine whether or not the methylation status of blood leukocyte DNA can be used as a surrogate marker of the risk for cancer, we quantitatively determined the methylation levels of insulin-like growth factor 2 (IGF2) and TUSC3 in 299 gastric cancer cases, and 299 age- and gender-matched controls. The IGF2 methylation levels in blood leukocyte DNA of the cases were lower than those of the healthy controls and there was a significant trend of increasing gastric cancer risk with decreasing methylation level of IGF2. Patients with hypermethylated IGF2 in blood leukocyte DNA showed a significantly better survival rate than those with hypomethylated IGF2, indicating that the IGF2 methylation level in blood leukocyte DNA can be a possible marker not only of the risk for but also of the prognosis of gastric cancer. In contrast, the TUSC3 methylation level in blood leukocyte DNA was higher in the cases than in the healthy controls, but the difference was not significant. The past lifestyle and clinicopathological characteristics of the participants were analyzed for any relationship with the methylation level. With aging and smoking, methylation of IGF2 and TUSC3 decreased and increased in blood leukocyte DNA, respectively. These results indicate that the methylation level of IGF2 in blood leukocyte DNA may be used as an important surrogate marker of the risk for gastric cancer.     

Mechanisms for the induction of gastric cancer by Helicobacter pylori infection: aberrant DNA methylation pathway

Multiple pathogenic mechanisms by which Helicobacter pylori infection induces gastric cancer have been established in the last two decades. In particular, aberrant DNA methylation is induced in multiple driver genes, which inactivates them. Methylation profiles in gastric cancer are associated with specific subtypes, such as microsatellite instability. Recent comprehensive and integrated analyses showed that many cancer-related pathways are more frequently altered by aberrant DNA methylation than by mutations. Aberrant DNA methylation can even be present in noncancerous gastric mucosae, producing an “epigenetic field for cancerization.” Mechanistically, H. pylori-induced chronic inflammation, but not H. pylori itself, plays a direct role in the induction of aberrant DNA methylation. The expression of three inflammation-related genes, Il1b, Nos2, and Tnf, is highly associated with the induction of aberrant DNA methylation. Importantly, the degree of accumulated aberrant DNA methylation is strongly correlated with gastric cancer risk. A recent multicenter prospective cohort study demonstrated the utility of epigenetic cancer risk diagnosis for metachronous gastric cancer. Suppression of aberrant DNA methylation by a demethylating agent was shown to inhibit gastric cancer development in an animal model. Induction of aberrant DNA methylation is the major pathway by which H. pylori infection induces gastric cancer, and this can be utilized for translational opportunities.

     

Blood leukocyte DNA hypomethylation and gastric cancer risk in a high‐risk Polish population

Global hypomethylation has been shown to increase genome instability potentially leading to increased cancer risk. We determined whether global methylation in blood leukocyte DNA was associated with gastric cancer in a population‐based study on 302 gastric cancer cases and 421 age‐ and sex‐matched controls in Warsaw, Poland, between 1994 and 1996. Using PCR‐pyrosequencing, we analyzed methylation levels of Alu and LINE‐1, 2 CG‐rich repetitive elements, to measure global methylation levels. Gastric cancer risk was highest among those with lowest level of methylation in either Alu (OR = 1.3, 95% CI = 0.9–1.9) or LINE‐1 (OR = 1.4, 95% CI = 0.9–2.0) relative to those with the highest levels, although the trends were not statistically significant. For Alu, the association was stronger among those aged 70 or older (OR = 2.6, 95% CI = 1.3–5.5, p for interaction = 0.02). We did not observe meaningful differences in the associations by other risk factors and polymorphisms examined. For LINE‐1, the association tended to be stronger among individuals with a family history of cancer (OR = 3.1, 95% CI = 1.4–7.0, p for interaction = 0.01), current alcohol drinkers (OR = 1.9, 95% CI = 1.0–3.6, p for interaction = 0.05), current smokers (OR = 2.3, 95% CI = 1.1–4.6, p for interaction = 0.02), those who rarely or never consumed fruit (OR = 3.1, 95% CI = 1.2–8.1, p for interaction = 0.03), CC carriers for the MTRR Ex5+123C>T polymorphism (OR = 2.3, 95% CI = 1.2–4.4, p for interaction = 0.01) and TT carriers for the MTRR Ex15+572T>C polymorphism (OR = 1.7, 95% CI = 1.0–2.8, p for interaction = 0.06). The association was not different by sex, Helicobacter pylori infection, intake of folate, vitamin B6 and total protein and the remaining polymorphisms examined. Our results indicate that interactions between blood leukocyte DNA hypomethylation and host characteristics may determine gastric cancer risk.     

基因芯片技术在膀胱癌研究中的应用

基因芯片技术是一种高通量的基因检测手段.基因芯片技术的出现,为人们研究膀胱癌发生发展过程中的基闪表达变化,特别是研究这一变化中的多基因、多途径的肿瘤演进过程的机制提供了强有力的工具.现综述近年来有关基因芯片技术在膀胱癌研究中的新进展.     

Discovery of differentially expressed genes in human breast cancer using subtracted cDNA libraries and cDNA microarrays

Identifying novel and known genes that are differentially expressed in breast cancer has important implications in understanding the biology of breast tumorigenesis and developing new diagnostic and therapeutic agents. In this study we have combined two powerful technologies, PCR-based cDNA subtraction and cDNA microarray, as a high throughput methodology designed to identify cDNA clones that are breast tumor- and tissue-specific and are overexpressed in breast tumors. Approximately 2000 cDNA clones generated from the subtracted breast tumor library were arrayed on the microarray chips. The arrayed target cDNAs were then hybridized with 30 pairs of fluorescent-labeled cDNA probes generated from breast tumors and normal tissues to determine the tissue distribution and tumor specificity. cDNA clones showing overexpression in breast tumors by microarray were further analysed by DNA sequencing, GenBank and EST database searches, and quantitative real time PCR. We identified several known genes, including mammaglobin, cytokeratin 19, fibronectin, and hair-specific type II keratin, which have previously been shown to be overexpressed in breast tumors and may play an important role in the malignance of breast. We also discovered B726P which appears to be an isoform of NY-BR-1, a breast tissue-specific gene. Two additional clones discovered, B709P and GABA(A) receptor pi subunit, were not previously described for their overexpression profile in breast tumors. Thus, combining PCR-based cDNA subtraction and cDNA microarray allowed for an efficient way to identify and validate genes with elevated mRNA expression levels in breast cancer that may potentially be involved in breast cancer progression. These differentially expressed genes may be of potential utility as therapeutic and diagnostic targets for breast cancer.     

Down-regulation of miR-212 expression by DNA hypermethylation in human gastric cancer cells

There has been few report discussing the expression and function of miR-212 in gastric cancer (GC). The aim of this pilot study was to investigate the expression of miR-212 in both gastric cancer tissues and gastric cancer cells and further explores the possible reasons for this change and the impact on the development of gastric cancer. qRT-PCR was used to detect the expression of miR-212 in primary GC tissues, adjacent normal tissues, gastric cancer cell lines BGC-823, SGC-7901, MKN-45, and normal gastric mucosa cell line GES. The expression of miR-212 was evaluated before and after treatment with methylation inhibitor-5-Aza-2'-deoxycitidine (5-Aza-dC), finally anti-miRNA and dual luciferase reporter assay were used to prove that MYC is a target gene of miR-212. The results showed that a significant reduction of miR-212 expression in GC tissues was observed compared to that in normal tissues (P = 0.002). At the same time, miR-212 expression level in normal gastric mucosa cell line GES was higher than that of in gastric cancer cell lines BGC-823, SGC-7901, and MKN-45 (P = 0.015, 0.008, 0.044, respectively). Computer sequence analysis showed the hypermethylation of CpG islands(CPI) in the promoter regions of miR-212 led to the lower expression of miR-212 in gastric cell strains (BGC-823 and SGC-7901). MiR-212 expression was significantly recovered after treatment with methylation inhibitor 5-Aza-dC (P = 0.016, 0.000, 0.015, respectively). Then, the results of AMOs transfection and dual luciferase reporter assay showed that Myc is a target of miR-212, which will be helpful to verify the function of miR-212 in carcinogenesis. The conclusion could be deduced from the study that decreased expression of miR-212 may be due to hypermethylation of CPI in gastric cancer cells, and miR-212 might act on the progression of gastric cancer through the potential target gene Myc.     

Mechanism of action of 5-AZA-dC: induced DNA hypomethylation does not lead to aberrant gene expression in human leukemic CEM cells

To understand the mechanism of action of 5-AZA-2'-deoxycytidine, a potent antineoplastic agent, we studied its effect in CEM cells on DNA methylation while monitoring coinciding changes in DNA, RNA and protein synthesis. At concentrations near the inhibitory concentration, the drug induced a profound reduction of DNA methylation. Effects on DNA and RNA synthesis were also noted at 24 and 48 h of treatment. However, at all concentrations assayed, no change in polypeptide composition as monitored by 2-D gel electrophoresis, were observed. That the cytotoxic effect of 5-AZA-deoxycytidine is due to changes in gene expression induced by DNA methylation reduction is not supported.