Abstract 2459 Epigenetic regulation of BRCA1 in breast cancer cells

BRCA1 is a tumor suppressor gene that plays an important role in DNA repair and replication, regulating double strand break repair through homologous recombination. Loss of functional BRCA1 activates error-prone repair pathways leading to mutations which cause cells to become cancerous. Mutations in BRCA1 are highly associated with breast cancer. Loss of BRCA1 can occur through epigenetic means, often through methylation of CpG islands in the promoter region, preventing transcription. Small non-coding RNA called microRNA (miRNA) are also capable of regulating BRCA1, which regulate gene expression after transcription by binding to mRNA. The purpose of this study was to explore the epigenetic regulation of BRCA1. To do this, we conducted experiments using the MCF-7 breast cancer cell line. We treated cells with 5μM and 10μM of 5-aza-cytidine for 72 hours, and 5nM siRNA for 48 hours targeting DNMT1 to investigate the change in BRCA1 after loss of DNA methylation. We extracted DNA and RNA from untreated and treated cells. To determine the levels of the BRCA1 gene, we carried out real time quantitative PCR using GAPDH as a reference. We also determine levels of miRNAs, using the Human miRNA CodeSet from NanoString. We carried out pyrosequencing to measure DNA methylation levels in the promoter of BRCA1. We found that expression of BRCA1 was 5x and 2x higher after exposure to 5-aza-cytidine (5μM, BRCA1 expression ratio=480.5%±309.6%) and DNMT1 silencing (BRCA1 expression ratio=231.2%±140.7%), suggesting loss of DNA methylation leads to an increase of expression of BRCA1. We investigated DNA methylation levels and found there was no change after exposure to 5μM 5-aza-cytidine or treatment with DNMT1 siRNA. Levels of DNA methylation were 7.7%±0.1% and 8.49%±0.0% in 5-aza-cytidine and DNMT1 siRNA treated cells, compared to 7.89%±0.1% in controls. These results suggest changes in expression of BRCA1 are not directly dependent on loss of DNA methylation in its promoter region. We then explored the regulation of BRCA1 expression by miRNAs. A total of 93 miRNAs were predicted to regulate BRCA1 using the informatic prediction softwares miRDB and miRTarbase. Of these, 10 miRNAs were deregulated in response to 5-aza-cytidine and DNMT1 siRNA treatment. Eight were underexpressed including miR-552-3p, miR-132-3p, miR-764, miR-575, miR-1297, miR-181a-5p, miR-186-5p, miR-26a-5p and miR-205-5p, suggesting a relationship exist between the miRNA level changes and the increase in BRCA1 expression. Our findings suggest epigenetic regulation of BRCA1 is complex, and miRNA deregulation through DNA methylation changes is mediated by other players. Our future research will focus on identifying these elements, to help us understand the mechanisms leading to the loss of BRCA1 in breast cancer in hopes of finding new therapeutic targets.