Abstract
BackgroundIt 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.MethodsLevels 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.ResultsNearly 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.ConclusionsOur 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.
Highlights
It has recently been proposed that the M-type phospholipase A2 receptor (PLA2R1) acts as a tumour suppressor in certain malignancies including mammary cancer
In normal cells and CAL-51 and UACC-812 cancer cell lines the PLA2R1 methylation degree was negligible (
The analysis revealed significantly increased levels of DNMT1 transcripts in MDAMB-453 cells with the highest PLA2R1 gene methylation followed by the BT-474 and MCF-7 cell lines (Fig. 3a)
Summary
It has recently been proposed that the M-type phospholipase A2 receptor (PLA2R1) acts as a tumour suppressor in certain malignancies including mammary cancer. M-type phospholipase A2 receptor (PLA2R1) is a 180 kDa transmembrane glycoprotein that belongs to the C-type lectin superfamily and the mannose receptor family. PLA2R1 consists of cystein-reach domain, fibronectin type II domain and eight carbohydrate recognition domains [1, 2]. The receptor binds secreted phospholipases A2 (sPLA2) with distinct affinities [3, 4]. As result of sPLA2 binding to PLA2R1, the amount of sPLA2 is lowered in extracellular milieu and its cellular signaling cascades linked to apoptosis and senescence are switched on [4]. Limited number of studies addressed the pathophysiological role of PLA2R1. It has been shown that PLA2R1 is the major podocyte autoantigen associated with development of idiopathic membranous nephropathy [5, 6]
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