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Gene expression analysis of extravillous trophoblast cells during the first-trimester

ABSTRACT: Purpose: During early placentation, extravillous trophoblast (EVT) cell invasion into maternal decidua plays a crucial role in establishment of the successful pregnancy. However, little is known about the characteristic features of EVT-associated ncRNAs. The purpose of this study is to elucidate gene expression profile for both coding and non-coding genes (i.e., mRNAs, lncRNAs, and miRNAs) expressed in EVT cells isolated from the first-trimester human placenta by RNA sequencing analysis. Methods: Human first trimester placental tissues (at gestational week 7, n=3) were obtained after legal abortions. EVT cells growing from explanted human chorionic villi were isolated. Minced chorionic villous tips were defined as first-trimester chorionic villous trophoblast (CVT) cells. Using total RNAs of these samples, mRNA/lncRNA-sequencing analysis was carried out on an Illumina MiSeq (San Diego, California, USA) using MiSeq v2 Reagent Kit 300cycle (cat. no. MS-102-2002; Illumina) and MiSeq v2 Reagent Kit 50cycle (cat. no. MS-102-2001; Illumina), respectively. The paired-end reads of 151 bp length for mRNA/lncRNA-sequencing and single reads of 26 bp length for miRNA-sequencing were obtained as fastq files which were imported into CLC genomics workbench (version 8.0.1; Qiagen, Venlo, Netherlands). Clean reads were aligned to the human genome sequence retrieved from the Ensembl database (Assembly GRCh38.p13, database version 82.38 and 101.38) and microRNA database miRbase (release 21), using CLC Genomics Workbench Map Reads to Reference tool. Normalization of gene expression data was performed by the TMM method of edgeR package. Statistically differentially expressed transcripts were conducted using edgeR. Genes that had a false discovery rate (FDR)-corrected p-value (q-value) of < 0.05 and an absolute log2 fold change (log FC) ≥ 1 were judged to be significantly differentially expressed (i.e., differentially expressed genes, DEGs). Results: Five thousand ninty-two mRNAs , 422 lncRNAs and 400 miRNAs were found to be differentially expressed. One hundred differentially expressed genes (i.e., 10 mRNAs, 7 lncRNAs, and 83 miRNAs) that are located in the chromosome 14q32.2 region containing the eutherian-specific imprinted DLK1-DIO3 domain were significantly down-regulated in EVT cells. In terms of imprinted genes (http://www.geneimprint.com/site/genes-by-species), 68 genes were found to be differentially expressed; 71% of these genes were down-regulated in EVT cells. lncRNA H19 was a DEG (q = 1.29.E-09, log FC = 2.98) and the most highly expressed lncRNA (70.47%) in EVT cells. Conclusions: The present research should be valuable for a resource for future ncRNA research in EVT cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE163651 | GEO | 2021/02/07

REPOSITORIES: GEO

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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 maternal and paternal copies of the genome are both required for mammalian development and this is primarily due to imprinted genes, those that are mono-allelically expressed based on parent-of-origin. Typically, this pattern of expression is regulated by differentially methylated regions (DMRs) that are established in the germline and maintained after fertilisation. There are a large number of germline DMRs that have not yet been associated with imprinting and their function in development is unknown. In this study, we developed a genome-wide approach to identify novel imprinted DMRs, specifically in the human placenta, and investigated the dynamics of imprinted DMRs during development in somatic and extra-embryonic tissues. DNA methylation was evaluated using the Illumina HumanMethylation450 array in 116 human tissue samples, publically available reduced representation bisulfite sequencing in the human embryo and germ cells, and targeted bisulfite sequencing in term placentas. 43 known and 101 novel imprinted DMRs were identified in the human placenta, by comparing methylation between diandric and digynic triploids and female and male gametes. 72 novel DMRs showed a pattern consistent with placental-specific imprinting and this mono-allelic methylation was entirely maternal in origin. Strikingly, these DMRs exhibited polymorphic imprinted methylation specifically in placenta. These data suggest that imprinting in human development is far more extensive and dynamic than previously reported and that the placenta preferentially maintains maternal germline-derived DNA methylation For the identification of imprinted DMRs in the placenta, chorionic villous samples from 5 diandric and 5 digynic triploids pregnancies were assayed, along with a pooled sample of complete hydatiform moles (CHM). Placental chorionic villous samples (n=63) included 29 control pregnancies delivered at term, while the remaining 34 were delivered preterm or miscarried, or had abnormal MSS results, IUGR or LOPET. The preterm births were associated with one or more of: preterm labour, premature rupture of membranes (PROM), chorioamnionitis, placental abruption, and incompetent cervix. All samples were determined to be chromosomally normal using standard karyotyping or comparative genome hybridization, as previously described (Robinson et al. 2010). Two to four independent sites were taken from each placenta and after DNA extraction from chorionic villous, the DNA was pooled before being utilized in this study. Thirty-three fetal tissues, including brain (n=8), spinal cord (n=7), muscle (n=9), and kidney (n=9) were collected from second trimester foetuses, as previously described (Price et al. 2012). Adult female whole blood samples (n=10) were collected from control women. Extra-embryonic cell types (n=19), including cord blood (embryonic), cord, amniotic membrane, chorionic membrane, 1st, 2nd and 3rd trimester trophoblast and mesenchyme, and decidua (maternal), were isolated from control placental samples.

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Gene expression analysis of extravillous trophoblast cells during the first-trimester

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