Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human trophoblast cell cultures

ABSTRACT: The syncytiotrophoblast develops as a result of the fusion of placental villous cytotrophoblast cells. These multinuclear cells are essential for fetal-maternal exchange and production of pregnancy-related hormones. Placental deficiency is considered a major risk factor associated with intrauterine growth restriction and preeclampsia. Better understanding of placental patho-physiology requires knowledge on gene expression regulation involved in trophoblast differentiation. Using the well-established in vitro trophoblast differentiation model, we have performed a microarray analysis on mRNA expression in trophoblast and syncytiotrophoblast cell cultures. Dramatic changes in gene expression patterns were detected during trophoblast differentiation. As many as 3524 novel and known genes are found to be up- or down regulated for more than 2-fold. Real-time PCR analysis of a group of genes confirmed the reliability of the microarray data. Overall, this study provided a global view on the molecular events underlying trophoblast differentiation. Subsequent characterization on regulation and function of the identified genes may lead to discovery of new pathways important for placental differentiation and function. Experiment Overall Design: Affymetrix GeneChipTM Human Genome U133 Plus 2.0 microarrays were used for mRNA profiling. RNA samples were subject to Agilent analysis for quality controls. cDNA was prepared from 10 Âµg of RNA, quantified by spectrometry, and used as a template for the synthesis of biotinylated cRNA using RNA transcript labeling reagent. The quality of the cRNA probe was verified by gel electrophoresis as well as a pilot hybridization using the Test-3 arrays. Hybridization solution containing fragmented cRNA probes and control cRNA was supplemented with herring sperm DNA and bovine serum albumin. The probe solution was heated at 99Â° C for 5 min followed by incubation at 45Â° C for 5 min before use. Hybridization was carried out at 45Â° C for 16 hr with constant rotation at 60 rpm. The arrays were washed and stained with streptavidin-phycoerythrin. The GeneChip 5.0 (Affymetrix) program was used to scan and quantitatively document the hybridization signals. Compilation of candidate gene and calculation of changes were performed on SpotFire and Microsoft Excel programs. To minimize the false-positive conclusion, only genes satisfying the following two criteria were considered candidate regulation targets for further analysis: 1) The geneâs hybridization signal reached an absolute level that was significantly higher than that of the background (P<0.05); 2) The changes is more than 2-fold when compared to control group.

ORGANISM(S): Homo sapiens

SUBMITTER: Jesus Gonzalez Bosquet

PROVIDER: E-GEOD-4100 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Estrogen-related receptor M-NM-3 (ERRM-NM-3) serves a critical O2-dependent regulatory role in differentiation of human cytotrophoblasts to syncytiotrophoblast. In this study, we investigated expression of genes encoding tissue kallikreins (KLK1) and voltage-gated K+ channels (KV7) during differentiation of human trophoblasts in culture and the roles of ERRM-NM-3 and O2 tension in their regulation. Expression of KLK1 and the KV7 channel subunits, KCNQ1, KCNE1, KCNE3, KCNE5, increased during differentiation of cultured human trophoblast cells in a 20% O2 environment. Notably, together with ERRM-NM-3, expression of KLK1, KCNQ1, KCNE1, KCNE3 and KCNE5 was markedly reduced when cells were cultured in a hypoxic environment (2% O2). Moreover, upon transduction of trophoblast cells with shRNAs for endogenous ERRM-NM-3, KLK1, KCNQ1, KCNE1 and KCNE3 expression was significantly decreased. Promoter and site-directed mutagenesis studies in transfected cells identified putative ERRM-NM-3 response elements (ERREs) within the KLK1 and KCNE1 5'-flanking regions required for ERRM-NM-3 -stimulated transcriptional activity. Binding of endogenous ERRM-NM-3 to these ERREs increased during trophoblast differentiation in culture and was inhibited by hypoxia. The KV7 blocker linopirdine reduced hCG secretion and aggregation of cultured human trophoblasts, suggesting a possible role of KV7 channels in cell fusion and differentiation. Illumina gene expression arrays of cultured human trophoblast cells revealed several genes upregulated during syncytiotrophoblast differentiation and downregulated upon ERRM-NM-3 knockdown involved in cell differentiation, adhesion, and synthesis of steroid and peptide hormones required for placental development and function. Collectively, these findings suggest that ERRM-NM-3 mediates O2-dependent expression of genes involved in human trophoblast differentiation, function and vascular homeostasis. Illumina whole genome gene expression array analysis was performed on human trophoblasts before culture, or 72 h after infection with recombinant lentiviruses expressing ERRM-NM-3 shRNA, or a control, nonsilencing shRNA. RNA from three independent experiments using placental tissues from three abortuses was extracted by miRNeasyM-BM-. Mini Kit (Qiagen, Maryland, USA). RNA was labeled and hybridized to an Illumina HumanHT-12 v4 BeadChip, according to the manufacturerM-bM-^@M-^Ys protocol.

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Transcription profiling of human trophoblast cell cultures

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