SP1 mediates the link between methylation of the tumour suppressor miR‐149 and outcome in colorectal cancer

Although recent studies indicate that DNA methylation contributes to the down‐regulation of microRNAs (miRNAs) in colorectal cancer (CRC), this field remains largely unexplored. To identify methylation‐silenced miRNAs and clarify their role in CRC, we performed a microarray analysis and screened for miRNAs that were induced in CRC cells by 5‐aza‐2′‐deoxycytidine treatment or by the knockdown of DNA methyltransferases. The DNA methylation status of the candidate miRNA was analysed by bisulphite sequencing PCR and methylation‐specific PCR. We found that miRNA‐149 (miR‐149) was epigenetically silenced in CRC and down‐regulation of miR‐149 was associated with hypermethylation of the neighbouring CpG island (CGI). Quantitative RT‐PCR analysis demonstrated that the miR‐149 level was markedly reduced in 51.6% of the CRC tissues compared with matched non‐cancerous tissues. In addition, low expression of miR‐149 was associated with a greater depth of invasion ($\it{p} = 0.012$ ), lower 5‐year survival rate ($\it{p} = 0.025$ ), and was found to be an independent prognostic factor for overall survival ($\it{p} = 0.016$ ) in a multivariate analysis. Moreover, transfection of miR‐149 inhibited cell growth and invasion of CRC cells in vitro. We also identified mRNA for Specificity Protein 1 (SP1, Sp1), a potential oncogenic protein, as a target of miR‐149. Our data suggest that, as a methylation‐sensitive miRNA, miR‐149 may play an important role as a tumour suppressor in CRC, which has prognostic and therapeutic implications. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.