Identification and Functional Analysis of Epigenetically Silenced MicroRNAs in Colorectal Cancer Cells

Abstract

Abnormal microRNA (miRNA) expression has been linked to the development and progression of several human cancers, and such dysregulation can result from aberrant DNA methylation. While a small number of miRNAs is known to be regulated by DNA methylation, we postulated that such epigenetic regulation is more prevalent. By combining MBD-isolated Genome Sequencing (MiGS) to evaluate genome-wide DNA methylation patterns and microarray analysis to determine miRNA expression levels, we systematically searched for candidate miRNAs regulated by DNA methylation in colorectal cancer cell lines. We found 64 miRNAs to be robustly methylated in HCT116 cells; eighteen of them were located in imprinting regions or already reported to be regulated by DNA methylation. For the remaining 46 miRNAs, expression levels of 18 were consistent with their DNA methylation status. Finally, 8 miRNAs were up-regulated by 5-aza-2′-deoxycytidine treatment and identified to be novel miRNAs regulated by DNA methylation. Moreover, we demonstrated the functional relevance of these epigenetically silenced miRNAs by ectopically expressing select candidates, which resulted in inhibition of growth and migration of cancer cells. In addition to reporting these findings, our study also provides a reliable, systematic strategy to identify DNA methylation-regulated miRNAs by combining DNA methylation profiles and expression data.

miR-1247 Functions by Targeting Cartilage Transcription Factor SOX9

“…ADAM15 has a predicted miR-1247-binding site in its 3Ј-UTR, and Yan and colleagues (40) suggest that the effect in migration, which they observed upon modulation of this miRNA could be mediated by its targeting ADAM15; however, they did not test this hypothesis. Although we detected subtle modulation of ADAM15 at the mRNA level upon overexpression and inhibition of miR-1247, this effect was not observed at the protein level and luciferase assays did not reveal any direct binding of miR-1247 to the 3Ј-UTR of ADAM15.…”

Genetic and epigenetic factors in regulation of microRNA in colorectal cancers

“…In colorectal cancer several miRNAs were found silenced by comparing primary colorectal cancers with matched normal colorectal epithelium [25]. In particular miR-129-2, miR-9-1, and miR-137 were found silenced by DNA methylation in cancer [26].…”

Unique DNA methylome profiles in CpG island methylator phenotype colon cancers

“…In fact, cap analysis of gene expression (CAGE) (Kodzius et al 2006) sequence tags were detected at significant proportions of differentially methylated non-promoter CpG islands in our study (Table 1), providing evidence of possible transcription initiation at these sites. Furthermore, DNA methylation can modulate miRNA expression (Han et al 2007;Lujambio et al 2007;Toyota et al 2008;Yan et al 2011) and binding of regulatory proteins, such as CCCTC-binding factor (CTCF) (Bell and Felsenfeld 2000;Yang et al 2003). Thus, nonpromoter differential methylation overlapping these genomic elements can also alter cellular functions through differential expression of miRNAs and regulation of enhancer activities (Supplemental Table 1).…”