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

J Pathol. 2013 Jan;229(1):12-24. doi: 10.1002/path.4078.


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 (p = 0.012), lower 5-year survival rate (p = 0.025), and was found to be an independent prognostic factor for overall survival (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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azacitidine / analogs & derivatives
  • Azacitidine / pharmacology
  • Base Sequence
  • Cell Proliferation
  • Chi-Square Distribution
  • Colorectal Neoplasms / genetics
  • Colorectal Neoplasms / metabolism*
  • Colorectal Neoplasms / mortality
  • Colorectal Neoplasms / pathology
  • CpG Islands* / drug effects
  • DNA Methylation* / drug effects
  • DNA Modification Methylases / antagonists & inhibitors
  • DNA Modification Methylases / genetics
  • DNA Modification Methylases / metabolism
  • Decitabine
  • Down-Regulation
  • Enzyme Inhibitors / pharmacology
  • Female
  • Gene Expression Profiling / methods
  • Gene Knockdown Techniques
  • Gene Silencing
  • HCT116 Cells
  • HT29 Cells
  • Humans
  • Kaplan-Meier Estimate
  • Male
  • MicroRNAs / metabolism*
  • Middle Aged
  • Molecular Sequence Data
  • Multivariate Analysis
  • Neoplasm Invasiveness
  • Oligonucleotide Array Sequence Analysis
  • Polymerase Chain Reaction
  • Prognosis
  • Proportional Hazards Models
  • Risk Assessment
  • Risk Factors
  • Signal Transduction
  • Sp1 Transcription Factor / genetics
  • Sp1 Transcription Factor / metabolism*
  • Time Factors
  • Transfection


  • Enzyme Inhibitors
  • MIRN149 microRNA, human
  • MicroRNAs
  • Sp1 Transcription Factor
  • Decitabine
  • DNA Modification Methylases
  • Azacitidine