Abstract 2401: Epigenetics-regulated microRNAs related with epithelial-mesenchymal transition of breast cancer cells
Authors: Sato Fumiaki , Ito Takaaki , Kawaguchi-Sakita Nobuko , Shimizu Kazuharu , Tsujimoto Gozoh , Toi Masakazu , Saji Shigehira 
Published: August 2012

Background: Epithelial-mesenchymal transition (EMT) is a program in which biological cells change morphologically and functionally from an epithelial phenotype to a mesenchymal phenotype. The EMT is involved in the process of cancer metastasis. On the other hand, accumulated evidence showed epigenetics and microRNA plays important roles in breast cancer. However, to date, biological networks between epigenetics and microRNAs regarding EMT remains largely unclear. Thus, the aim of this study is to identify epigenetics-regulated microRNAs related with EMT of breast cancer. Materials / Methods and Results: MicroRNA expression profiles of 11 breast cancer cell lines (8 epithelial-phenotype (E-type) cells and 3 mesenchymal-phenotype (M-type) cells) were obtained by microarray method. Unsupervised clustering analysis showed that E- and M-type breast cancer cells had different microRNA expression profiles. On the other hand, we obtained a genomewide methylation status of these breast cancer cell lines by a MeDIP-seq method. An integrated in silico analysis identified microRNAs which microRNA and DNA methylation were inversely correlated. All of miR-200b/a/429 cluster members were listed in top5 differentially expressed miRs between E- and M-type cells, and also in top 5 epigenetics-regulated microRNAs. In the further study, we focused upon the miR-200b/a/429 cluster. A COBRA assay revealed that promoter regions of miR-200b/a/429 cluster in M-type breast cancer cells were more frequently methylated than that in E-type cells (65.1% vs 6.8%, p&lt;0.0001, respectively). The methylation levels were inversely associated with miR-200b/a/429 cluster expression (Taqman assay, p&lt;0.01). In addition, demethylating treatment using 5-aza-dC unmasked miR-200b/a/429 expression in M-typed breast cancer cell lines. Taken together, the finding indicated that the expression of miR-200b/a/429 cluster is epigenetically regulated. Next, we investigated an effect of the miR-200b/a/429 cluster upon cell motility as a function of the EMT. A transfection of exogenous miR-200b/429 inhibited 24% of cell migration ability (Transwell assay). Utilizing microRNA target prediction algorithm, we identified fibronectin as a target gene of miR-200b/429. Utilizing several prediction algorithms for microRNA target genes, we identified fibronectin as a target gene of miR-200b/429. A luciferase-based reporter assay demonstrated that miR-200b/429 were directly associated with fibronectin-3′UTR and repressed 21% (p&lt;0.0001) of the reporter gene expression post-transcriptionally. Conclusion: The promoter hypermethylation of miR-200b cluster is associated with epithelial-mesenchymal transition, and stimulates cell motility by upregulating fibronectin expression in mesenchymal-phenotype breast cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2401. doi:1538-7445.AM2012-2401