Abstract Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that some miRNAs are silenced in association with CpG island hypermethylation in cancer. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by 5-aza-2′-deoxycytidine (DAC) and 4-phenylburtyrate (PBA) treatment in GC cell lines. By using miRNA microarray (Agilent technologies), we analyzed a panel of 470 miRNAs in MKN74 and AGS cells treated with or without DAC and PBA, and found that 172 and 151 of the miRNAs were significantly upregulated (>5-fold) by the drug treatment, respectively. Among them, we noted miR-34b and miR-34c, which are direct targets of p53, and are frequently silenced in colorectal cancer. Here, we show that miR-34b and -c are frequently downregulated in GC in association with DNA hypermethylation. Reporter assay revealed strong promoter activity in the upstream CpG island of miR-34b/c. Methylation of the CpG island was frequently observed in GC cell lines (13/13, 100%) and primary GC specimens (83/118, 70%), but not in normal gastric mucosa. By in situ hybridization analysis, we observed downregulated expression of miR-34b in primary GC specimens. Methylation of the CpG island was also detected in the non-tumorous gastric mucosa of the GC patients. Notably, quantitative methylation analysis by bisulfite-pyrosequencing revealed higher methylation levels in gastric mucosa with multiple GCs than those with single GCs. Transfection of precursor molecules of miR-34b or miR-34c into GC cells suppressed cellular growth. Gene expression microarray analysis revealed that miR-34b or miR-34c induced dramatic changes in the gene expression profile in GC cells. Particularly, “cell cycle” and “M phase” genes are significantly enriched among the genes downregulated by miR-34b/c. Our results suggest that miR-34b/c are novel tumor suppressors in GC, and that methylation of miR-34b/c CpG island might be a useful marker to predict GC risk. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4945.