ABSTRACT
GPER1, also known as GPR30, is a novel seven-transmembrane G-protein coupled estrogen receptor that mediates both short-term (non-genomic) and long-term (genomic) effects of estrogen in target cells and tissues. A substantial body of work over the last two decades has highlighted its therapeutic or prognostic utility. However, the clinical data on the expression of GPER1 in breast tissue is ambiguous. Analysis of TCGA RNAseq data revealed significantly lower mean expression of GPER1 mRNA in primary breast tumors compared to that in normal breast tissues. This provides support to the tumor suppressor role for GPER1. However, the mechanisms underlying the reduced expression are not completely understood. We analyzed the expression levels of GPER1 mRNA variants in MCF-7 and MDA-MB-231 cells by RT-PCR, and the methylation status of two CpG islands in the GPER1 locus by modified COBRA assays and bisulfite sequencing. Our results show that MCF-7 cells express higher levels of GPER1 mRNA variants compared to MDA-MB-231 cells. Modified COBRA assays revealed differential methylation in the upstream CpG island (upCpGi) that overlaps with the first exon of two GPER1 variants (GPER1v2 and v3) but not in the downstream CpG island (dnCpGi) that overlaps with the coding region common to all variants. Bisulfite sequencing results showed that the core upCpGi was hypo-methylated in both MCF-7 and MDA-MB-231 cells. However, eight CpGs in the 3' end of the upCpGi were hyper-methylated in MDA-MB-231 cells. 5-Azacytidine, a DNA methyltransferase inhibitor, induced the expression levels of GPER1 mRNA variants in MDA-MB-231 cells. Expression-methylation correlation analysis of TCGA breast cancer data revealed that methylation of CpGs in the regions flanking the upCpGi significantly correlated negatively with GPER1 mRNA expression. Taken together, our results demonstrate the role of DNA methylation in GPER1 repression, implicate the flanking regions (shore) of the upCpGi, and suggest a potential mechanism of GPER1 silencing in breast tumors.
Subject(s)
Breast Neoplasms/genetics , CpG Islands/genetics , DNA Methylation , Gene Silencing , Receptors, Estrogen/genetics , Receptors, G-Protein-Coupled/genetics , Azacitidine/pharmacology , Breast Neoplasms/pathology , Cell Line, Tumor , DNA, Neoplasm/chemistry , DNA, Neoplasm/genetics , Enzyme Inhibitors/pharmacology , Exons/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Genetic Variation , Humans , MCF-7 Cells , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA/methodsABSTRACT
Rabbit antiserum was generated against the N-terminus of human GPR30 followed by peptide affinity purification. In this article, the methodology used and validation data are presented. The peptide affinity purified polyclonal antibody specifically detects human GPR30 in ELISA and on western blots of total protein prepared from human breast cancer cell lines.
