Accumulation of aberrant DNA methylation in normal-appearing gastric mucosae, mostly induced by H. pylori infection, is now known to be deeply involved in predisposition to gastric cancers (epigenetic field defect), and silencing of protein-coding genes has been analyzed so far. In this study, we aimed to clarify the involvement of microRNA (miRNA) gene silencing in the field defect. First, we selected three miRNA genes as methylation-silenced after analysis of six candidate ''methylation-silenced'' tumor-suppressor miRNA genes. Methylation levels of the three genes (miR124a-1, miR-124a-2 and miR-124a-3) were quantified in 56 normal gastric mucosae of healthy volunteers (28 volunteers with H. pylori and 28 without), 45 noncancerous gastric mucosae of gastric cancer patients (29 patients with H. pylori and 16 without), and 28 gastric cancer tissues (13 intestinal and 15 diffuse types). Among the healthy volunteers, individuals with H. pylori had 7.8-13.1-fold higher methylation levels than those without (p < 0.001). Among individuals without H. pylori, noncancerous gastric mucosae of gastric cancer patients had 7.2-15.5-fold higher methylation levels than gastric mucosae of healthy volunteers (p < 0.005). Different from protein-coding genes, individuals with past H. pylori infection retained similar methylation levels to those with current infection. In cancer tissues, methylation levels were highly variable, and no difference was observed between intestinal and diffuse histological types. This strongly indicated that methylationsilencing of miRNA genes, in addition to that of protein-coding genes, contributed to the formation of a field defect for gastric cancers. ' 2008 Wiley-Liss, Inc.Key words: field for cancerization; microRNA; methylation; gastric cancer; Helicobacter pylori Metachronous occurrence of gastric cancers is becoming an important issue as localized resection of early gastric cancers by endoscopic submucosal dissection (ESD) has become common. 1 The incidence of secondary primary gastric cancers after ESD reaches as high as 2.0% per year 2 whereas the incidence of gastric cancer in the general Japanese population is 0.14% per year. 3 This indicates that noncancerous gastric mucosae are already predisposed to developing gastric cancers, forming a field defect (field for cancerization). High incidences of metachronous cancers have been known not only for gastric cancers but also for bladder, liver, and esophageal cancers 4-6 and are becoming recognized for lung, breast and colorectal cancers. [7][8][9] A molecular basis for the field defect has been considered as an accumulation of genetic and epigenetic alterations in normalappearing tissues. Traditionally, cells with a genetic alteration were considered to form a physically continuous patch, producing a genetically altered field. 10 Recently, we found that aberrant DNA methylation of specific genes can be induced in as high as several percentage of cells in noncancerous gastric mucosae (thus in multiple independent gastric glands), and the degre...

BackgroundEpigenetic alterations accumulate in normal-appearing tissues of patients with cancer, producing an epigenetic field defect. Cross-sectional studies show that the degree of the defect may be associated with risk in some types of cancer, especially cancers associated with chronic inflammation.ObjectiveTo demonstrate, by a multicentre prospective cohort study, that the risk of metachronous gastric cancer after endoscopic resection (ER) can be predicted by assessment of the epigenetic field defect using methylation levels.DesignPatients with early gastric cancer, aged 40–80 years, who planned to have, or had undergone, ER, were enrolled at least 6 months after Helicobacter pylori infection discontinued. Methylation levels of three preselected genes (miR-124a-3, EMX1 and NKX6-1) were measured by quantitative methylation-specific PCR. Patients were followed up annually by endoscopy, and the primary endpoint was defined as detection of a metachronous gastric cancer. Authentic metachronous gastric cancers were defined as cancers excluding those detected within 1 year after the enrolment.ResultsAmong 826 patients enrolled, 782 patients had at least one follow-up, with a median follow-up of 2.97 years. Authentic metachronous gastric cancers developed in 66 patients: 29, 16 and 21 patients at 1–2, 2–3 and ≥3 years after the enrolment, respectively. The highest quartile of the miR-124a-3 methylation level had a significant univariate HR (95% CI) (2.17 (1.07 to 4.41); p=0.032) and a multivariate-adjusted HR (2.30 (1.03 to 5.10); p=0.042) of developing authentic metachronous gastric cancers. Similar trends were seen for EMX1 and NKX6-1.ConclusionsAssessment of the degree of an epigenetic field defect is a promising cancer risk marker that takes account of life history.

ABSTRACT

A multiplex analysis for profiling the expression of candidate genes along with epigenetic modification may lead to a better understanding of the complex machinery of neuropathic pain. In the present study, we found that partial sciatic nerve ligation most remarkably increased the expression of monocyte chemotactic protein 3 (MCP-3, known as CCL7) a total of 33 541 genes in the spinal cord, which lasted for 4 weeks. This increase in MCP-3 gene transcription was accompanied by the decreased trimethylation of histone H3 at Lys27 at the MCP-3 promoter. The increased MCP-3 expression associated with its epigenetic modification observed in the spinal cord was almost abolished in interleukin 6 knockout mice with partial sciatic nerve ligation. Consistent with these findings, a single intrathecal injection of recombinant proteins of interleukin 6 significantly increased MCP-3 messenger RNA with a decrease in the level of Lys27 trimethylation of histone H3 at the MCP-3 promoter in the spinal cord of mice. Furthermore, deletion of the C-C chemokine receptor type 2 (CCR2) gene, which encodes a receptor for MCP-3, failed to affect the acceleration of MCP-3 expression in the spinal cord after partial sciatic nerve ligation. A robust increase in MCP-3 protein, which lasted for up to 2 weeks after surgery, in the dorsal horn of the spinal cord of mice with partial sciatic nerve ligation was seen mostly in astrocytes, but not microglia or neurons. On the other hand, the increases in both microglia and astrocytes in the spinal cord by partial sciatic nerve ligation were mostly abolished in interleukin 6 knockout mice. Moreover, this increase in microglia was almost abolished by CCR2 gene deletion, whereas the increase in astrocytes was not affected in nerve-ligated mice that lacked the CCR2 gene. We also found that either in vivo or in vitro treatment with MCP-3 caused robust microglia activation. Under these conditions, intrathecal administration of MCP-3 antibody suppressed the increase in microglia within the mouse spinal cord and neuropathic pain-like behaviours after nerve injury. With the use of a functional magnetic resonance imaging analysis, we demonstrated that a single intrathecal injection of MCP-3 induced dramatic increases in signal intensity in pain-related brain regions. These findings suggest that increased MCP-3 expression associated with interleukin 6 dependent epigenetic modification at the MCP-3 promoter after nerve injury, mostly in spinal astrocytes, may serve to facilitate astrocyte-microglia interaction in the spinal cord and could play a critical role in the neuropathic pain-like state.