Epigenetic regulation of miR-34b and miR-129 expression in gastric cancer Kuo-Wang Tsai 1 , Chew-Wun Wu 2 , Ling-Yueh Hu 3 , Sung-Chou Li 3,4,5 , Yu-Lun Liao 3 , Chun-Hung Lai 3 , Hsiao-Wei Kao 3 , Wen-Liang Fang 2,6 , Kuo-Hung Huang 2,6 , Wen-Ching Chan 3,4,5 and Wen-Chang Lin 3,4 1 Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, Republic of China 2 Department of Surgery, Veterans General Hospital, Taipei, Taiwan, Republic of China 3 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China 4 Institute of Biomedical Informatics, National Yang-Ming University, Taipei, Taiwan, Republic of China 5 Bioinformatics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan, Republic of China 6 Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China MicroRNAs (miRNAs) are small noncoding RNAs that play fundamental roles in diverse biological and pathological processes by targeting the expression of specific genes. Here, we identified 38 methylation-associated miRNAs, the expression of which could be epigenetically restored by cotreatment with 5-aza-2 0 -deoxycytidine and trichostatin A. Among these 38 miRNAs, we further analyzed miR-34b, miR-127-3p, miR-129-3p and miR-409 because CpG islands are predicted adjacent to them. The methylation-silenced expression of these miRNAs could be reactivated in gastric cancer cells by treatment with demethylating drugs in a time-dependent manner. Analysis of the methylation status of these miRNAs showed that the upstream CpG-rich regions of mir-34b and mir-129-2 are frequently methylated in gastric cancer tissues compared to adjacent normal tissues, and their methylation status correlated inversely with their expression patterns. The expression of miR-34b and miR-129-3p was downregulated by DNA hypermethylation in primary gastric cancers, and the low expression was associated with poor clinicopathological features. In summary, our study shows that tumor-specific methylation silences miR-34b and miR-129 in gastric cancer cells. Gastric cancer is a common type of cancer in humans, espe- cially in the Andean region of South America and the Far East. The clinical outcomes for patients with gastric cancer are poor, with a 5-year survival rate of only 20%, and only about 40% of patients respond to surgical intervention. 1 Most patients have advanced gastric cancer at diagnosis, resulting in a poor survival rate. Therefore, clarifying the mechanisms underlying the cancer in detail and improving diagnostic sen- sitivity in its early stages should improve the survival rate in gastric cancer patients. MicroRNAs (miRNAs) are naturally occurring, very small, noncoding RNAs that are involved in the regulation of gene expression by inducing mRNA degradation or repressing mRNA translation. 2 They are typically 18–25 nucleotides in length and are processed from large hairpin precursors, which are encoded widely in the genomes of animals and plants. miRNAs are critical in biological functions such as cell proliferation, apoptosis, neuron development and tumori- genesis. 3–5 They can function as tumor suppressors or onco- genes, depending on the target gene. Tumor-suppressor miR- NAs usually repress growth-promoting genes and are downregulated in cancers. Conversely, oncogenic miRNAs target cell-growth-inhibiting genes, and their expression is of- ten upregulated in cancers. 6 The dysregulation of miRNAs can be caused by various mechanisms, including deletions, amplifications or mutations of the genomic DNA as well as aberrant epigenetic modulation. Epigenetic modification involves DNA methylation and the covalent modification of histone proteins and does not directly change the DNA sequence. DNA methylation is a heritable and enzyme- induced modification in humans. A high frequency of CpG dinucleotides is often found in the 5 0 -untranslated regions and first exons of most genes, and these dinucleotides control the expression of these genes by modulating their methyla- tion status. 7 The methylation of CpG islands in the gene promoter has been strongly linked to the silencing of tumor-suppressor gene expression in cancer. 6,8–12 In gastric cancer cells, the hy- permethylation of CpG islands in the promoter region has Key words: miR-34b, miR-129, tumor-specific methylation, CpG islands, 5-Aza-dC Additional Supporting Information may be found in the online version of this article. Grant sponsor: Center of Excellence for Cancer Research at Taipei Veterans General Hospital; Grant number: DOH99-TD-C-111-007; Grant sponsor: Academia Sinica DOI: 10.1002/ijc.25919 History: Received 15 Jun 2010; Accepted 10 Dec 2010; Online 6 Jan 2011 Correspondence to: Wen-Chang Lin, Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, Republic of China, Tel.: þ(886)-2-2652-3967, Fax: þ(886)-2-2782-7654, E-mail: wenlin@ibms.sinica.edu.tw Cancer Genetics Int. J. Cancer: 129, 2600–2610 (2011) V C 2011 UICC International Journal of Cancer IJC