Effect of CpG island methylation on microRNA expression in the k-562 cell line.

To test the hypothesis that methylation of a CpG island is associated with regulation of microRNA expression, we investigated CpG islands in the upstream sequences of microRNA precursors (pre-miRNAs) through bioinformatic analysis and determined whether the CpG islands were methylated by methylation-specific PCR in the k-562 cell line. We used 5-azacytidine for DNA demethylation, and changes in microRNA expression were detected by microarray assay, RT-PCR, and real-time PCR after 5-azacytidine induction. We showed that the CpG islands in the upstream regions of 18 pre-miRNAs were methylated, including miR-663, miR-369, miR-615, and miR-410, and promoter activity was detected in the upstream region of pre-miR-663. We found that a decrease in methylation of a CpG island could up-regulate the expression of miR-663, suggesting that miR-663 could be regulated by DNA methylation. Expression levels of miR-369, miR-615, and miR-410 were not regulated by DNA methylation in this cell line.