Early-onset preeclampsia, plasma microRNAs and endothelial cell function.

Abstract

BACKGROUND\nPreeclampsia is a hypertensive pregnancy disorder, in which generalized systemic inflammation and maternal endothelial dysfunction are involved in the pathophysiology. MiRNAs are small non-coding RNAs responsible for post-transcriptional regulation of gene expression and involved in many physiological processes. They mainly downregulate translation of their target genes.\n\n\nOBJECTIVE\nWe aimed to compare the plasma miRNA concentrations in preeclampsia, healthy pregnancy and non-pregnant women. Furthermore, we aimed to evaluate the effect of three highly increased plasma miRNAs in preeclampsia on endothelial cell function in vitro.\n\n\nSTUDY DESIGN\nWe compared 3,391 (precursor) miRNA concentrations in plasma samples from early-onset preeclamptic women, gestational age matched healthy pregnant women and non-pregnant women using miRNA 3.1. arrays (Affymetrix) and validated our findings by real-time quantitative PCR (RT qPCR). Subsequently, endothelial cells (human umbilical vein endothelial cells) were transfected with microRNA mimics (we choose the three miRNAs with the highest fold change and lowest false discovery rate in preeclampsia vs. healthy pregnancy). After transfection, functional assays were performed to evaluate if overexpression of the microRNAs in endothelial cells affected endothelial cell function in vitro. Functional assays were the wound healing assay (which measures cell migration and proliferation), the proliferation assay and the tube formation assay (which assesses formation of endothelial cell tubes during the angiogenic process). To determine if the miRNAs are able to decrease gene expression of certain genes, RNA was isolated from transfected endothelial cells and gene expression (by measuring RNA expression) was evaluated by gene expression microarray (Genechip Human Gene 2.1 ST arrays [Life Technologies]). For the microarray we used pooled samples, but the differently expressed genes in the microarray were validated by RT qPCR in individual samples.\n\n\nRESULTS\nNo significant differences (fold change < -1.2 or > 1.2 with a false discovery rate < 0.05) were found in miRNA plasma concentrations between healthy pregnant and non-pregnant women. The plasma concentrations of 26 (precursor) miRNAs were different between preeclampsia and healthy pregnancy. The 3 miRNAs which were increased with the highest fold change and lowest false discovery rate in preeclampsia vs. healthy pregnancy were miR-574-5p, miR-1972, and miR-4793-3p. Transfection of endothelial cells with these miRNAs in showed that miR-574-5p decreased (p<0.05) the wound healing capacity (i.e. decreased endothelial cell migration and/or proliferation) and tended (p<0.1) to decrease proliferation, miR-1972 decreased tube formation (p<0.05) and also tended (p<0.1) to decrease proliferation and miR-4793-3p tended (p<0.1) to decrease both the wound healing capacity and tube formation in vitro. Gene expression analysis of transfected endothelial cells revealed that miR-574-5p tended (p<0.1) to decrease the expression of the proliferation marker MKI67.\n\n\nCONCLUSION\nWe conclude that in the early-onset preeclampsia group in our study different concentrations of plasma miRNAs are present as compared with healthy pregnancy. Our results suggest that miR-574-5p and miR-1972 decrease the proliferation (probably via decreasing MKI67) and/or migration as well as the tube formation capacity of endothelial cells. Therefore, these miRNAs may be anti-angiogenic factors affecting endothelial cells in preeclampsia.