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

Ae-jin Choi,Hongli Yan,Angela H. Ting,Axel Imhof,Byron H. Lee

doi:10.1371/journal.pone.0020628

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.

Highlights

MicroRNAs are small, non-coding RNAs that regulate gene expression and play pivotal roles in normal cellular processes including proliferation, differentiation, and apoptosis [1]
Gene promoter DNA methylation is negatively correlated to gene expression and can account for aberrant tumor suppressor gene silencing in a variety of human cancers [7]
Double Knockout (DKO) cells are HCT116 cells deleted for DNA methyltransferase 21 and 23b and retain,5% genomic DNA methylation [20]

Summary

Introduction

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and play pivotal roles in normal cellular processes including proliferation, differentiation, and apoptosis [1]. Silenced miRNAs have been discovered in cancers based on differential expression between normal tissues and tumors or between baseline and DNA demethylated cancer cells. Using 1.5-fold change as our threshold, we identified 18 miRNAs as having consistent expression data with their DNA methylation status in HCT116 and DKO cells (Table S1).

Results

Conclusion