Elsevier Trophoblast Research Award Lecture: origin, evolution and future of placenta miRNAs.

MicroRNAs (miRNAs) regulate the expression of a large number of genes in plants and animals. Placental miRNAs appeared late in evolution and can be found only in mammals. Nevertheless, these miRNAs are constantly under evolutionary pressure. As a consequence, miRNA sequences and their mRNA targets may differ between species, and some miRNAs can only be found in humans. Their expression can be tissue- or cell-specific and can vary time-dependently. Human placenta tissue exhibits a specific miRNA expression pattern that dynamically changes during pregnancy and is reflected in the maternal plasma. Some placental miRNAs are involved in or associated with major pregnancy disorders, such as preeclampsia, intrauterine growth restriction or preterm delivery and, therefore, have a strong potential for usage as sensitive and specific biomarkers. In this review we summarize current knowledge on the origin of placental miRNAs, their expression in humans with special regard to trophoblast cells, interspecies differences, and their future as biomarkers. It can be concluded that animal models for human reproduction have a different panel of miRNAs and targets, and can only partly reflect or predict the situation in humans.

IFPA Meeting 2012 Workshop Report II: epigenetics and imprinting in the placenta, growth factors and villous trophoblast differentiation, role of the placenta in regulating fetal exposure to xenobiotics during pregnancy, infection and the placenta.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 there were twelve themed workshops, four of which are summarized in this report. These workshops related to various aspects of placental biology: 1) epigenetics and imprinting in the placenta; 2) growth factors and villous trophoblast differentiation; 3) role of the placenta in regulating fetal exposure to xenobiotics during pregnancy; 4) infection and the placenta.

MicroRNA expression profiles of trophoblastic cells.

BACKGROUND: MicroRNAs (miRNAs) are small single-stranded RNA molecules working as post-transcriptional modulators of gene expression. Trophoblast cells are a heterogenous group of fetal cells forming the feto-maternal interface and displaying a wide spectrum of functions. The regulation of their behavior may partly underly the control through miRNAs. Therefore, we aimed to compare the miRNA profile of primary first and third trimester trophoblast cells with that of different trophoblastic cell lines. MATERIAL AND METHODS: Total RNA was obtained from isolated cytotrophoblast cells from healthy term and first trimester placentae and the cell lines HTR-8/SVneo (immortalized trophoblast cells), JEG-3 (choriocarcinoma), ACH-3P and AC1-M59, which are choriocarcinoma cells fused with first and third trimester trophoblast cells, respectively. The expression level of 762 different miRNAs was quantitatively analyzed by using a TaqMan Human MicroRNA Array. For testing the reproducibility of the array technique, the expression of 9 selected miRNAs has been re-analyzed by individual qPCR. RESULTS: The analyzed cell types share many similar patterns of miRNAs, but are significantly distinct in the expression of three miRNA clusters: chromosome 19 miRNA cluster (C19MC; containing 54 different miRNAs), C14MC (34 miRNAs) and a minor cluster (miRNA-371 to miRNA-373 cluster), also located on chromosome 19. Expression of miRNAs within C19MC increases significantly from first to third trimester trophoblast while that of C14MC members decreases. MiRNAs within the miR-371-3 cluster augment slightly. C19MC and the miR-371-3 cluster are not expressed by HTR-8/SVneo cells whilst C14MC is almost not detectable in the choriocarcinoma-derived cell lines complete array data available at NCBI Gene Expression Omnibus accession number GSE32346: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32346). Beside the miRNAs within the mentioned clusters, further 27 miRNAs are differentially expressed (>100 fold) between term and first trimester trophoblast cells. The placenta-specific miRNAs miR-141 and miR-21 as well as let-7g are expressed in all tested cells with the highest expression in primary trophoblast cells. CONCLUSION: Primary first trimester and term trophoblast cells and trophoblastic cell lines display major differences in their miRNA fingerprints which may be involved in their different behavior and characteristics.

PP007. Effects of STAT1 suppression on ERK1/2 in trophoblastic cells.

INTRODUCTION: Migration and trophoblast invasion are controlled functionally along with the active participation of cytokines and growth factors. Two important intracellular signaling pathways are the Janus kinase/signal transducer and activator of transcription (JAK-STAT) and extracellular regulated kinase1/2 (ERK1/2). These pathways have been associated with the regulation of gene expression, cellular proliferation, differentiation, angiogenesis, embryo development and invasion in tumor and trophoblast cells. OBJECTIVES: The aim of our study is to characterize and analyze the regulation and crosstalks of STAT1 and ERK1/2 in trophoblast cells and the identification of activating cytokines. METHODS: The trophoblast derived cell line HTR-8/svneo and a choriocarcinoma cell line (JEG-3) were stimulated with interleukin-6 (IL-6), IL-11, granulocyte-macrophage colony-stimulating factor (GMC-SF), leukemia inhibitory factor (LIF) or oncostatine M (OSM). The the expression and phosphorylation of STAT1(tyr705) and ERK1/2 were analyzed by gel electrophoresis and Western blotting. Expression of STAT1 was inhibited by administration of 50µM fludarabine (2-fluoro-ara-AMP) for 2, 4, 8, 24, 48 or 72h or by using small interfering RNA (siRNA). The full activation of STAT1 was assessed by using an STAT1 DNA-binding assay. Finally, proliferation and invasion assays were performed (Grant Deutscher Akademischer Austausch Dienst A/10172477). RESULTS: LIF and OSM induce STAT1 and ERK1/2 phosphorylation in HTR-8 and JEG-3 cells. Fludarabine inhibits the so induced phosphorylation of STAT1 when administered 48 or 72h before stimulation. Simultaneously, ERK phosphorylation increases. In contrast, silencing of STAT1 by application of specific siRNA induces reduction of ERK1/2 phosphorylation. Fludarabine reduces STAT1 DNA-binding capacity. LIF and OSM increase proliferation. Silencing of STAT1 slightly decreases invasiveness of analyzed cells. CONCLUSION: STAT1 in trophoblast cells can be activated by placental cytokines. Suppression of STAT1 by fludarabine or siRNA influences activity of ERK1/2 which indicates a crosstalk between both pathways. Current studies will clarify the reason for the different effects on ERK1/2 in trophoblastic cells.