Methylation-associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect

Abstract

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.

Aberrant Methylation of RASGRF1 Is Associated with an Epigenetic Field Defect and Increased Risk of Gastric Cancer

“…We previously showed hypomethylation of LINE-1 repetitive elements and hypermethylation of CDH1 in enlarged-fold gastritis, which is an indicator of a high risk for diffuse type gastric cancer (8). More recently, we and others reported frequent hypermethylation of tumor suppressive miRNA genes in the noncancerous gastric mucosa of patients with gastric cancer (9,10). Taken together, these results suggest that aberrant methylation in the gastric mucosa could be a useful biomarker for evaluating gastric cancer risk.…”

“…Gastric cancer cell lines (MKN7, SH101, SNU1, SNU638, JRST, KatoIII, AZ521, AGS, and NCI-N87) were obtained and cultured as described previously (9,13). SH101 and HSC43 cells were kindly provided by Dr. Kazuyoshi Yanagihara, Yasuda Women's University (14,15).…”

“…Genomic DNA (1 mg) was modified with sodium bisulfite using an EpiTect Bisulfite Kit (QIAGEN) after which methylation-specific PCR (MSP), bisulfite sequencing, and pyrosequencing were carried out as described previously (9). Briefly, for bisulfite pyrosequencing, the biotinylated PCR product was purified, made single-stranded, and used as a template in a pyrosequencing reaction run according to the manufacturer's instructions.…”

“…Western blot analysis was carried out as described previously (9). Mouse anti-V5 monoclonal antibody (mAb; Invitrogen), rabbit anti-RASGRF1 polyclonal Ab (sc-863, Santa Cruz Biotechnology), and mouse antiactin mAb (Chemicon) were used as instructed by the manufacturers.…”

Inflammation-induced repression of tumor suppressor miR-7 in gastric tumor cells

“…Helicobacter pylori infection induces chronic gastritis, resulting in induction of DNA methylation (Niwa et al, 2010). The expression of miR-34b/c is suppressed by DNA methylation in H. pylori-associated gastric cancer cells (Suzuki et al, 2010). Moreover, H3K27me3 leads to tumorsuppressor gene silencing in cancer (Kondo et al, 2008).…”