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Epigenetic Silencing of MicroRNA-34b/c and B-Cell Translocation Gene 4 Is Associated with CpG Island Methylation in Colorectal Cancer

¹ First Department of Internal Medicine and ² Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University, Sapporo, Japan

Requests for reprints: Hiromu Suzuki, First Department of Internal Medicine, Sapporo Medical University, S1, W16, Chuo-Ku, Sapporo, 060-8543, Japan. Phone: 81-11-611-2111, ext. 3211; Fax: 81-11-618-3313; E-mail: hsuzuki{at}sapmed.ac.jp.

Key Words: colorectal cancer • microRNA • methylation • epigenetics • p53

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that, in cancer, expression of some miRNAs cells is silenced in association with CpG island hypermethylation. To identify epigenetically silenced miRNAs in colorectal cancer (CRC), we screened for miRNAs induced in CRC cells by 5-aza-2'-deoxycytidine (DAC) treatment or DNA methyltransferase knockout. We found that miRNA-34b (miR-34b) and miR-34c, two components of the p53 network, are epigenetically silenced in CRC; that this down-regulation of miR-34b/c is associated with hypermethylation of the neighboring CpG island; and that DAC treatment rapidly restores miR-34b/c expression. Methylation of the miR-34b/c CpG island was frequently observed in CRC cell lines (nine of nine, 100%) and in primary CRC tumors (101 of 111, 90%), but not in normal colonic mucosa. Transfection of precursor miR-34b or miR-34c into CRC cells induced dramatic changes in the gene expression profile, and there was significant overlap between the genes down-regulated by miR-34b/c and those down-regulated by DAC. We also found that the miR-34b/c CpG island is a bidirectional promoter which drives expression of both miR-34b/c and B-cell translocation gene 4 (BTG4); that methylation of the CpG island is also associated with transcriptional silencing of BTG4; and that ectopic expression of BTG4 suppresses colony formation by CRC cells. Our results suggest that miR-34b/c and BTG4 are novel tumor suppressors in CRC and that the miR-34b/c CpG island, which bidirectionally regulates miR-34b/c and BTG4, is a frequent target of epigenetic silencing in CRC. [Cancer Res 2008;68(11):4123–32]