Simone V Lip1, Mark V Boekschoten2, Guido J Hooiveld2, Mariëlle G van Pampus3, Sicco A Scherjon3, Torsten Plösch3, Marijke M Faas4. 1. Department of Obstetrics and Gynaecology, University Medical Center Groningen, University of Groningen, Groningen. Electronic address: S.V.Lip@umcg.nl. 2. Department of Nutrition, Metabolism and Genomics Group, Wageningen University, Wageningen, the Netherlands. 3. Department of Obstetrics and Gynaecology, University Medical Center Groningen, University of Groningen, Groningen. 4. Department of Pathology and Medical Biology, Division of Medical Biology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.
Abstract
BACKGROUND: Preeclampsia is a hypertensive pregnancy disorder in which generalized systemic inflammation and maternal endothelial dysfunction are involved in the pathophysiology. MiRNAs are small noncoding RNAs responsible for post-transcriptional regulation of gene expression and involved in many physiological processes. They mainly downregulate translation of their target genes. OBJECTIVE: We aimed to compare the plasma miRNA concentrations in preeclampsia, healthy pregnant women, and nonpregnant women. Furthermore, we aimed to evaluate the effect of 3 highly increased plasma miRNAs in preeclampsia on endothelial cell function in vitro. STUDY DESIGN: We compared 3391 (precursor) miRNA concentrations in plasma samples from early-onset preeclamptic women, gestational age-matched healthy pregnant women, and nonpregnant women using miRNA 3.1. arrays (Affymetrix) and validated our findings by real-time quantitative polymerase chain reaction. Subsequently, endothelial cells (human umbilical vein endothelial cells) were transfected with microRNA mimics (we choose the 3 miRNAs with the greatest fold change and lowest false-discovery rate in preeclampsia vs healthy pregnancy). After transfection, functional assays were performed to evaluate whether 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 whether 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 real-time quantitative polymerase chain reaction in individual samples. RESULTS: No 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 nonpregnant women. The plasma concentrations of 26 (precursor) miRNAs were different between preeclampsia and healthy pregnancy. The 3 miRNAs that were increased with the greatest 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<.05) the wound-healing capacity (ie, decreased endothelial cell migration and/or proliferation) and tended (P<.1) to decrease proliferation, miR-1972 decreased tube formation (P<.05), and also tended (P<.1) to decrease proliferation, and miR-4793-3p tended (P<.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<.1) to decrease the expression of the proliferation marker MKI67. CONCLUSION: We 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 antiangiogenic factors affecting endothelial cells in preeclampsia.
BACKGROUND: Preeclampsia is a hypertensive pregnancy disorder in which generalized systemic inflammation and maternal endothelial dysfunction are involved in the pathophysiology. MiRNAs are small noncoding RNAs responsible for post-transcriptional regulation of gene expression and involved in many physiological processes. They mainly downregulate translation of their target genes. OBJECTIVE: We aimed to compare the plasma miRNA concentrations in preeclampsia, healthy pregnant women, and nonpregnant women. Furthermore, we aimed to evaluate the effect of 3 highly increased plasma miRNAs in preeclampsia on endothelial cell function in vitro. STUDY DESIGN: We compared 3391 (precursor) miRNA concentrations in plasma samples from early-onset preeclamptic women, gestational age-matched healthy pregnant women, and nonpregnant women using miRNA 3.1. arrays (Affymetrix) and validated our findings by real-time quantitative polymerase chain reaction. Subsequently, endothelial cells (human umbilical vein endothelial cells) were transfected with microRNA mimics (we choose the 3 miRNAs with the greatest fold change and lowest false-discovery rate in preeclampsia vs healthy pregnancy). After transfection, functional assays were performed to evaluate whether 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 whether 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 real-time quantitative polymerase chain reaction in individual samples. RESULTS: No 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 nonpregnant women. The plasma concentrations of 26 (precursor) miRNAs were different between preeclampsia and healthy pregnancy. The 3 miRNAs that were increased with the greatest 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<.05) the wound-healing capacity (ie, decreased endothelial cell migration and/or proliferation) and tended (P<.1) to decrease proliferation, miR-1972 decreased tube formation (P<.05), and also tended (P<.1) to decrease proliferation, and miR-4793-3p tended (P<.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<.1) to decrease the expression of the proliferation marker MKI67. CONCLUSION: We 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 antiangiogenic factors affecting endothelial cells in preeclampsia.