Upregulation of the miR-515 cluster in the MCF7 breast cancer cell line by epigenetic therapy

Introduction. In cancer, hypermethylation of promoter CpG islands has been shown to down regulate the transcription of tumor suppressor genes. Treatment by inhibitors of DNA methyltransferease (DNMTi) can reactivate the transcription of silenced genes and restore normal cell growth and differentiation. Recent investigations show that the expression of microRNAs (miRs) with tumor suppressor properties may also be down regulated by epigenetic mechanisms in cancer. Aims. To investigate if miRs are down regulated by DNA methylation in the MCF7 breast cancer cell line. Methods. MCF7 cells were cultured in MEM medium with 10% FBS. The cells were treated with 1 mu M of the DNMTi 5-aza-2'-deoxycytidine (5-Aza-CdR) for 72 hours. Total RNA was extracted using Trizol, and 250 ng was labelled and hybridised to Exiqon miRCURY 9.2 arrays according to manufactures protocol in a dyeswap setup. Arrays were scanned using a GenePix 4200B scanner, and converted to intensity values using GenePix 6.1. Subsequently, data were analysed using R 2.6.1 and the Limma package from bioconductor. Intensity values were background corrected using an adaptive background correction (normexp). Normalization was done using LOESS and differential expression was calculated using linear models implemented in Limma and corrected for multiple testing using BH transformation. Results. Treatment with 5-Aza-CdR showed that 20 out of 576 ( similar to 3%) human miRs examined were significantly upregulated in the MCF7 cell line. Interestingly, 14 of these belonged to the miR-515 cluster on chromosome 19q13. Upregulation of the miRs was confimed by real time Q-PCR. According to Gene Bank, a CpG island is located just upstream of the cluster. Further analyses. We are currently performing 5' rapid amplification of cDNA ends (5'-RACE) to identify the transcription start site in order to investigate its actual methylation status. Potential oncogenic miR targets including NRAS, MAPK3, CCND1, AKT1 and EGFR were identified using database search. The mRNA and protein expression levels in treated vs. untreated cells will be investigated.