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Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells – implications for complications of pregnancy

ABSTRACT: Cell-to-cell communication is essential for the spiral artery (SpA) remodeling and required for maternal transfer of nutrient and oxygen to the fetus. Extravillous trophoblast invade the uterine arteries to remodel the SpA during the first 20 weeks of gestation. Interestingly, oxygen tenstion in the uterine environment changes with the progression of pregnancies and damage in the SpA remodelling can lead to preeclampsia and pther pregnancy complications. Recently, our understanding of how cells communicate has undergone a paradigm shift since the recognition of the role of exosomes on intercellular signaling. Here, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT), modifying their bioactivity on endothelial cells. Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™) was used to establish the effect of exosomes on endothelial cells. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly hifger in EVT culturedunder 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, while exosomes from 1% oxygen decrease EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of uniques miRNA in exosomes from EVT cultured at 1% oxygen and exosomes isolated from maternal at early gestation, who developed PE and SPTB later in pregnancy. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically useful biomarker of pregnancy complications.

ORGANISM(S): Homo sapiens

PROVIDER: GSE93020 | GEO | 2017/06/14

SECONDARY ACCESSION(S): PRJNA359418

REPOSITORIES: GEO

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Project description:Exososmes, potent intercellular communicators, are supposed to contribute to metastasis formation, which we confirmed for exosomes of the metastatic rat pancreatic adenocarcinoma line BSp73ASML that promote metastatic settlement in lymph nodes and lung of poorly metastatic BSp73ASML cells with a selective CD44v4-v7 (BSp73ASML-CD44vkd) knockdown. To define the molecular pathway(s), whereby exosomes contribute to premetastatic niche preparation, we profiled mRNA miRNA of BSp73ASMLwt and BSp73ASML-CD44vkd- exosomes and evaluated the impact on potential target cells. BSp73ASML exosomes are recovered in the draining lymph node after subcutaneous injection. In vitro, they preferentially bind and are taken-up by lymph node stroma cells (LnStr) and lung fibroblasts (LuFb) that were chosen as exosome targets. BSp73ASMLwt and BSp73ASML-CD44kd exosomes contain a restricted repertoire of mRNA and miRNA, hwere the lattter differe significantly between the two lines and even more pronounced, exosomes derived thereof with a not yet explored dominance of tumor-suppressor miRNA in ASML-CD44kd cells and exosomes. Both, exosomal mRNA and miRNA are recovered in target cells and exosome-uptake is accompanied by significant changes in gene expression. We didn't observe a correlation between exosomal mRNA and changes in target cell mRNA or proteins. Instead transferred miRNA significantly affected target cell mRNA translation as demonstrated for selected, most abundant ASML exosomal miRNA besides others, miR-494 known target MAL (myelin and lymphocytes protein)/cadherin17, and miR-542-3p which targets TRAF/cadherin17. Furthermore, MMP transcription suggested to accompany cadherin17 dwon-regulation was upregulated in miR-494 or miR542-3p transfected or exosome co-cultured LnStr. Taken together, tumor exosomes target in vivo non-transformed cells in premetastatic organs. Exosome uptake induced altered target celll gene expression is strongly promoted by exosomal miRNA where we demonstrate for the first time that exosomes/exosomal miRNA from a metastasizing tumor line can modulate stroma cells from premetastatic organs. Endothelial cells lines were treated with pancreatic adenocarcinoma (AS) derived exosomes or pancreatic adenocarcinoma derived exosomes expressing tetraspanin 8. Total RNA was isolated and used to perform the Agilent gene expression microarrays. In this assay a replicate of endothelial cell lines treated with ASTspan8 were also included. Moreover, total RNA from both base line expression of endothelial cells and rat endothelial fibroblasts were also used to perfrom gene expression microarrays. RNA isolated from Rat endothelial fibroblasts treated with the exosomes derived from rat pancreatic adenocarcinoma and exosomes derived from rat pancreatic adenocarcinoma expressing tetraspanin8 were individually used to perfrom gene expression microarrays. RNA isolated from exosomes derived from rat pancreatic adenocarcinoma cell lines expressing tetraspanin were used to peform gene expresiion to see the base line expression. Another replicate were also used. RNA isolated from base line or control of rat pancreatic adenocarcinoma wild type cells and also base line RNA isolated from rat pancreatic adenocarcinoma cells lines where CD44 was knock-down.

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Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells – implications for complications of pregnancy

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