title: Epigenetic control of phospholipase A2 receptor expression in mammary cancer cells.
abstract: BACKGROUND
  It has recently been proposed that the M-type phospholipase A2 receptor (PLA2R1) acts as a tumour suppressor in certain malignancies including mammary cancer. Considering that DNA methylation is an important regulator of gene transcription during carcinogenesis, in the current study we analyzed the PLA2R1 expression, PLA2R1 promoter methylation, and selected micro RNA (miRNA) levels in normal human mammary epithelial cells (HMEC) and cancer cell lines.
  
  
  METHODS
  Levels of PLA2R1 and DNA methyltransferases (DNMT) specific mRNA were determined using real-time RT-PCR. Methylation specific-high resolution melting (MS-HRM) analysis was utilized to quantify the methylation degree of selected CpG sites localized in the promoter region of the PLA2R1 gene. Expression of miRNA was tested using miScript Primer Assay system.
  
  
  RESULTS
  Nearly complete methylation of the analyzed PLA2R1 promoter region along with PLA2R1 gene silencing was identified in MDA-MB-453 mammary cancer cells. In MCF-7 and BT-474 mammary cancer cell lines, a higher DNA methylation degree and reduced PLA2R1 expression were found in comparison with those in normal HMEC. Synergistic effects of demethylating agent (5-aza-2'-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on PLA2R1 transcription in MDA-MB-453 cells confirmed the importance of DNA methylation and histone modification in the regulation of the PLA2R1 gene expression in mammary cells. Furthermore, significant positive correlation between the expression of DNMT1 and PLA2R1 gene methylation and negative correlation between the cellular levels of hsa-mir-141, -181b, and -181d-1 and the expression of PLA2R1 were identified in the analyzed cells. Analysis of combined z-score of miR-23b, -154 and -302d demonstrated a strong and significant positive correlation with PLA2R1 expression.
  
  
  CONCLUSIONS
  Our data indicate that (i) PLA2R1 expression in breast cancer cells is controlled by DNA methylation and histone modifications, (ii) hypermethylation of the PLA2R1 promoter region is associated with up-regulation of DNMT1, and (iii) hsa-miR-23b, -154, and -302d, as well as hsa-miR-141, -181b, and -181d-1 are potential candidates for post-transcriptional regulation of PLA2R1 expression in mammary cancer cells.
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty "Carl Gustav Carus", Technical University of Dresden, Fetscherstr. 74, 01307, Dresden, Germany. Mario.Menschikowski@uniklinikum-drersden.de.
authors.forename: Mario
authors.initials: M
authors.lastname: Menschikowski
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty "Carl Gustav Carus", Technical University of Dresden, Fetscherstr. 74, 01307, Dresden, Germany. Albert.Hagelgans@uniklinikum-drersden.de.
authors.forename: Albert
authors.initials: A
authors.lastname: Hagelgans
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty "Carl Gustav Carus", Technical University of Dresden, Fetscherstr. 74, 01307, Dresden, Germany. Brit.Nacke@uniklinikum-drersden.de.
authors.forename: Brit
authors.initials: B
authors.lastname: Nacke
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty "Carl Gustav Carus", Technical University of Dresden, Fetscherstr. 74, 01307, Dresden, Germany. Carsten.Jandeck@uniklinikum-drersden.de.
authors.forename: Carsten
authors.initials: C
authors.lastname: Jandeck
authors.affiliation: School of Health Sciences, Flinders University of South Australia, Bedford Park, SA, 5042, Australia. olga.sukocheva@flinders.edu.au.
authors.forename: Olga
authors.initials: O
authors.lastname: Sukocheva
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty "Carl Gustav Carus", Technical University of Dresden, Fetscherstr. 74, 01307, Dresden, Germany. Gabriele.Siegert@uniklinikum-drersden.de.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Siegert
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D054507:Receptors, Phospholipase A2 / Q000235:genetics*
chemical_list: D035683:MicroRNAs; C516360:PLA2R1 protein, human; D054507:Receptors, Phospholipase A2