Hypoxia induces trophoblast migration and invasion gene pathways, a subset of which are reduced in preeclampsia
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ABSTRACT: Placentation requires the proper regulation of extravillous trophoblast (EVT) migration and invasion into the decidua and maternal vasculature, processes which are initiated in physiologic hypoxic conditions. Abnormal EVT migration and/or invasion have been suggested to lead to pregnancy complications, such as preeclampsia. The objectives of this study are to determine how exposure to hypoxia impacts gene expression and cellular motility of first trimester trophoblasts, and to assess if expression of migration-associated genes is dysregulated in 2nd trimester chorionic villous samples (CVS) from preeclampsia pregnancies relative to CVS from healthy pregnancies. The 1st trimester trophoblast cell line, HTR8/SVneo, was used to investigate the relationship between hypoxia and Notch signaling in trophoblast migration and invasion. RNA sequencing and quantitative RT-PCR analyses show that exposure to hypoxia (2.5% O2) activates Notch signaling in HTR-8/SVneo. We demonstrate that exposure of HTR-8/SVneo to hypoxia induces expression of genes associated with cellular migration and invasion and increases HTR-8/SVneo cellular migration and invasion, whereas inhibition of gamma-secretase decreases Notch signaling and decreases HTR-8/SVneo migration and invasion. Analysis of RNA sequencing data from CVS of preeclampsia and uncomplicated pregnancies identified significant differentially expressed genes that are involved in cellular migration and invasion. Decreased expression of migration and invasion genes in CVS from preeclampsia pregnancies, may impair trophoblast migration and invasion in the 2nd trimester of pregnancy, resulting in the development of preeclampsia.
Project description:Preeclampsia is characterized by insufficient implantation of trophoblasts. It is relevant in the failure adaptation of trophoblasts to changes in the intrauterine embryo development environment. Fewer miRNAs have been reported on this. MiR-455-5p expressed lowly in preeclampsia but its role remains unknown. Combining cell and molecular biology methods, we suggested the function and relevant mechanisms of miR-455-5p and its potential target Shc3 in preeclampsia. In vitro, HTR-8/SVneo migrated and invaded more rapidly under H/R than in hypoxic or normoxic conditions when miR-455-5p was overexpressed. And the apoptosis of HTR-8/SVneo is in contrast to the migration and invasion in H/R. Shc3 was identified as a direct downstream target gene of miR-455-5p, where overexpression of this gene reversed the miR-455-5p impact, promoting apoptosis and suppressed invasion and migration of HTR-8/SVneo under H/R. Shc3 was highly expressed in H/R, but its level was low in isolated hypoxic or normoxic environments. Further, Shc3 overexpression is involved in placental inflammation and angiogenesis inhibition. Finally, we got the conclusion that the downregulation of miR-455-5p in preeclampsia contributes to the elevation of Shc3 in trophoblast, thereby deteriorating trophoblast cells implantation. The elevated Shc3 is associated with placental inflammation and angiogenesis inhibition. Shc3 serves as a potential biomarker for preeclampsia diagnosis and treatment.
Project description:Chronical hypoxia is a common occurrence following reduced uteroplacental blood flow resulting from incomplete trophoblast invasion and abnormal vascular remodeling of the spiral arteries in PE. Hypoxia can lead to active glycolysis, which increases the production of lactate, a substrate for histone lactylation. To screen for genes that may be regulated by histone lactylation in PE placentas, we performed RNA-seq in HTR-8/SVneo cells under hypoxia or treated with sodium L-lactate. The results showed that 3578 genes were upregulated in the HTR-8/SVneo cells under hypoxia compared to those under normoxia, and 355 genes were upregulated in HTR-8/SVneo cells treated with sodium L-lactate compared to the control cells. 152 upregulated genes in the HTR-8/SVneo cells under hypoxia and those treated with sodium L-lactate overlapping.
Project description:Aberrantexpression of long non-coding RNAs (lncRNAs) has been reported to play a crucial role in the biological function of trophoblasts and contribute to preeclampsia (PE). Our previous study demonstrated that MIR193BHG is increased in preeclamptic placental tissues. In this study, we further explored the effects of MIR193BHG on the function of trophoblast cells and attempted to elucidate its underlying molecular mechanisms. Results showed that MIR193BHG was predominantly localized in the nucleus of HTR-8/SVneo cells. Overexpression of MIR193BHG significantly inhibited proliferation, migration and invasion of HTR-8/SVneo cells, while increasing apoptosis. Knockdown of MIR193BHG produced opposite effects.Furthermore, overexpression of MIR193BHG led to significant increases in both mRNA and protein levels of p53 compared with the control group. More importantly, knockdown of p53 rescued the negative effects induced by overexpressed MIR193BHG on cell proliferation, migration, and invasion while partially counteracting its apoptotic effects on HTR-8/SVneo cells. In conclusion, our findings suggest that MIR193BHG played a critical role in the progression of PE by regulating the expression of p53 and may serve as a novel therapeutic target for PE.
Project description:Reduced or absent cytotrophoblast invasion of the maternal uterine spiral arteries is a common clinical finding in studies of pregnancies complicated by preeclampsia, suggesting that the mechanisms behind invasion of these cells is perturbed. The placenta initially develops in a low oxygen environment of 1-2% oxygen until after the 10th week of pregnancy. During this time oxygen concentration exerts a major influence over trophoblast activity and, in vitro, hypoxia inducible factors are proposed to be one of many key regulators of first trimester trophoblast behaviour. We used a global gene expression microarray approach to identify signalling pathways involved in invasion of the first trimester trophoblast cell line HTR8/SVneo under hypoxic conditions where HIF-1 was active. Additionally, first trimester placental samples from different gestational age groups were labelled with anti HIF-1 and HIF-2 to evaluate whether HIFs are differentially expressed and localised across the period of development characterised by hypoxia (6-8 weeks) and maternal blood perfusion (10-12 weeks). Eighty-eight genes were differentially expressed between cells cultured in 1% oxygen (where HIF-1 was localised to the nucleus) and 5% oxygen (where HIF-1 was cytoplasmic). 65% of the genes were predicted to contain HIF-1α:ARNT transcription factor binding sites. Increased nuclear localisation of HIF-1α was seen in extravillous cytotrophoblasts in early first trimester compared with late, while cellular expression of HIF-2α in the villous stroma was higher in late first trimester. While HIFs and their downstream targets are clearly induced in trophoblasts during early placental development, and in vitro hypoxic conditions, the mechanism and pathways by which invasion is increased under hypoxic conditions is not clear from the gene expression profile. Further insight beyond the transcription level is required to fully understand this complex phenomenon.
Project description:Invasion of cytotrophoblasts into uterine tissues is essential for placental development. To identify molecules regulating trophoblast invasion, mRNA signatures of purified villous (CTB, poor invasiveness) and extravillous (EVT, high invasiveness) trophoblasts isolated from first trimester human placentae and villous explant cultures, respectively, were compared using GeneChip analyses yielding 991 invasion/migration related transcripts. Several genes involved in physiological and pathologic cell invasion, including ADAM-12,-19,-28 as well as Spondin-2, were upregulated in EVT. Pathway prediction analyses identified several functional modules associated with either the invasive or the non-invasive trophoblast phenotype. One of the genes which were downregulated in the invasive mRNA pool, heme oxygenase-1 (HO-1), was selected for functional analyses. Real-time PCR analyses, Western blottting, and immunofluorescene of first trimester placentae and differentiating villous explant cultures demonstrated downregulation of HO-1 in invasive EVT as compared to CTB. Modulation of HO-1 expression in loss-of as well as gain-of function cell models (BeWo and HTR8/SVneo, respectively) demonstrated an inverse relationship of HO-1 expression with trophoblast migration in transwell and wound healing assays. Importantly, HO-1 expression led to an increase in protein levels and activity of the nuclear hormone receptor PPARgamma. Pharmacological inhibition of PPARgamma abrogated the inhibitory effects of HO-1 on trophoblast migration. Collectively, our results demonstrate that gene expression profiling of EVT and CTB can be used to unravel novel regulators of cell invasion. Accordingly, we identify heme oxygenase-1 as a negative regulator of trophoblast motility acting via upregulation of PPARgamma. Experiment Overall Design: To identify genes potentially regulating cell invasion trophoblast cells of early human gestation with distinct invasive properties were profiled. Experiment Overall Design: Distinct gene expression signatures of highly invasive EVT (n = 6) and poorly invasive CTB (n = 5) of different first trimester placentae using Affymetrix U133A GeneChips interrogating >20,000 genes were determined.
Project description:The goal of this study is to compare mRNAs expressed by EGF treated HTR-8/SVneo cells to iRNAs expressed in untreated control HTR-8/SVneo cells to identify various genes which play a role during EGF-mediated HTR-8/SVneo cell invasion
Project description:The goal of this study is to compare miRNAs expressed by EGF treated HTR-8/SVneo cells to miRNAs expressed in untreated control HTR-8/SVneo cells to identify micro RNAs which play a role during EGF-mediated HTR-8/SVneo cell invasion
Project description:The goal of this study is to compare miRNAs expressed by HGF treated HTR-8/SVneo cells to miRNAs expressed in untreated control HTR-8/SVneo cells to identify micro RNAs which play a role during HGF-mediated HTR-8/SVneo cells invasion
Project description:The goal of this study is to compare genes expressed by IFN-gamma treated HTR-8/SVneo cells to genes expressed in untreated control HTR-8/SVneo cells to identify genes which play a role during IFN-gamma-mediated HTR-8/SVneo cells invasion
Project description:The goal of this study is to compare miRNAs expressed by IFN-gamma treated HTR-8/SVneo cells to miRNAs expressed in untreated control HTR-8/SVneo cells to identify micro RNAs which play a role during IFN-gamma-mediated HTR-8/SVneo cells invasion