The Assembly of miRNA-mRNA-protein Regulatory Networks Using High-throughput Expression Data (miRNA)
ABSTRACT: Analyze miRNA expression levels in primary trophoblasts, derived from term human placenta and cultured under standard or hypoxic conditions Human placental trophoblasts were dispersed using a trypsin-deoxyribonuclease-dispase/Percoll method, plated in 6-well plates, and maintained in standard culture conditions (O2=20%). After 4 h (defined as time 0), the plates were divided to those in continued standard culture conditions, or to culture in hypoxia (O2=0%). Cells were then harvested at 6 h, 12 h, 24 h, 48 h and 72 h, and processed for miRNA arrays
Project description:Analyze gene expression levels in primary trophoblasts, derived from term human placenta and cultured under standard or hypoxic conditions Human placental trophoblasts were dispersed using a trypsin-deoxyribonuclease-dispase/Percoll method, plated in 6-well plates, and maintained in standard culture conditions (O2=20%). After 4 h (defined as time 0), the plates were divided to those in continued standard culture conditions, or to culture in hypoxia (O2=0%). Cells were then harvested at 6 h, 12 h, 24 h, 48 h and 72 h, and processed for mRNA arrays
Project description:We hypothesized altered expression of Proteases in calls capable of physiological invasion vs angiogenesis. We analyzed trophoblasts isolated from first trimester placenta that are invasive, and placental endothelial cells, that gave a high angiogenic potential. We found different expression levels of most proteases. We found that the expression of proteases differes in cells performing invasion vs cells performing angiogenesis. Trophoblasts were isolated from first trimester placenta, endothelial cells were isolated from third trimester placenta. A pool of 5 preparations from different cell types was used for each microarray.
Project description:YY1 is a sequence-specific DNA-binding transcription factor that has many important biological roles. However, its function in trophoblasts at the maternal-foetal interface remains to be elucidated. In this study, we used an mRNA microarray and quantitative reverse transcription-PCR and compared the YY1 mRNA expression level in trophoblasts between patients with recurrent miscarriage (RM) and healthy control subjects. Our results revealed that YY1 mRNA expression was significantly lower in the trophoblasts of the RM group compared with the healthy control group. Furthermore, immunofluorescence and immunohistochemical data showed that YY1 was highly expressed in human placental villi during early pregnancy, especially in cytotrophoblast cells and invasive extravillous trophoblasts, and it was expressed at a much lower level in the placental villi of term pregnancy. YY1 overexpression enhanced the invasion and proliferation of trophoblasts, while knockdown of YY1 repressed these effects. Antibody array screening revealed that YY1 significantly promoted MMP2 expression in trophoblasts. Bioinformatics analysis identified three YY1-binding sites in the MMP2 promoter region, and chromatin immunoprecipitation analysis verified that YY1 binds directly to its promoter region. Importantly, inhibition of YY1 by siRNA clearly decreased trophoblast invasion in an ex vivo explant culture model. Overall, our findings revealed a new regulatory pathway of YY1/MMP2 in trophoblast cells invasion during early pregnancy, and indicated that YY1 may be involved in the pathogenesis of RM. Total RNA was isolated using Trizol from trophoblast cells from three healthy controls (HC) and three recurrent miscarriage (RM) patients. Total RNA were extracted and used for hybridizing Affymetrix chips (GeneChip® Human Transcriptome Array 2.0(HTA2.0)). Data were normalised by gcRMA method and raw p-values adjusted by Bonferroni procedure (1%).
Project description:Analyze miRNA expression levels in HTR-8/SVeo cells stably transfected with a BAC plasmid containing the entire C19MC miRNA cluster HTR-8/SVeo cells were transfected with a modified BAC plasmid containing the entire C19MC miRNA cluster and carrying a zeocin selection cassette. Several independent clones and a mixed population of transfected cells were analyzed and compared to non-transfected HTR-8/SVeo cells and primary human trophoblasts that express the C19MC miRNAs endogenously
Project description:We compare two groups of transcriptome profiles. They are 1) primary human trophoblasts (PHT) and 2) syncytiotrophoblast from human pluripotent stem cells. This data set represents the former data set and the latter has been deposited separately at GSE72712. Primary human trophoblasts (PHT) were purified cytotrophoblast populations from whole term placentas and then cultured in vitro. The cytotrophoblasts were cultured for short (9 h) or long (48 h) term that show undifferentiated (PHTu) or differentiated (PHTd) phenotype that led syncytialization, respectively. Transcriptome profiles of PHTu and PHTd were compared with three size fractioned syncytiotrophoblasts and trophoblasts from human pluripotent stem cells that are named ESCd_gt70 (greater than 70 µm), ESCd_40-70 (size between 40 and 70 µm), ESCd_lt40 (smaller than 40 µm) and undifferentiated progenitor (ESCu; see GSE72712). Primary human trophoblasts (PHT) were derived and cultured from three term human placentas obtained by the Obstetrical Specimen Procurement Unit from women after a healthy pregnancy, labor, and delivery at Magee-Womens Hospital of the University of Pittsburgh Medical Center. Isolated PHT lines obtained from three individual (1 female and 2 male) placentas were cultured separately.
Project description:Primary term human trophoblasts were derived from placentas after a healthy pregnancy, and exposed to ionizing irradiation (vs sham) in vitro Primary human trophoblasts were irradiated 24 h after initial plating, defined as time zero. Cells were irradiated at 10 Gy using a Clinac 600C (Varian Medical Systems, Palo Alto, CA) with a 6 MV photon beam and a dose rate of 250 cGy/min. The flasks containing the cells were placed on 1.5 cm of bolus (a tissue equivalent material) since the maximum irradiation depth was 1.5 cm, which corresponded to the plated cell layer. Cells were analyzed 4, 8, and 24 h after irradiation or sham.
Project description:P. falciparum NF54 proliferates under micro-aerophilic conditions in an environment of 3% O2, 4% CO2, 93% N2. This strain was gradually adapted to proliferate under standard tissue culture conditions of 5% CO2/95% air (~19% O2) to generate P. falciparum HOX. We compared global gene expression profiles of the two strains to identify differences, if any. Asynchronous cultures of P. falciparum NF54 and HOX propagated in O+ RBCs were processed and gene expression analyzed on Affymetrix microarrays. All cultures consised of 80% rings + trophozoites.
Project description:Endothelial cells, and many other types of cells too, physiologically reside in low O2 environments (~ 2-13% O2 or 15-60 mmHg pO2; [PCN]) relevantly to atmospheric O2 (~ 21% or 160 mmHg pO2 at sea level) in vivo. Such PCN is critical for endothelial functions. The majority of our current knowledge regarding the cellular and signaling mechanisms governing endothelial functions, however, is built on endothelial models established under atmospheric O2 (~21% O2). Herein, we comapred the transcriptional profiles between HUVE and HUAE cells cultured and expanded under PCN (3% oxygen) and standard culture normoxia (21% O2). We established human umbilical vein (HUVE) and artery (HUAE) endothelial cell cultures under PCN (3% O2; 20-25 days) and SCN (21% O2), and examined the global gene expression using Affymetrix U133 plus 2.0 microarray chips.
Project description:Estrogen-related receptor γ (ERRγ) 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 ERRγ 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 ERRγ, 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 ERRγ, KLK1, KCNQ1, KCNE1 and KCNE3 expression was significantly decreased. Promoter and site-directed mutagenesis studies in transfected cells identified putative ERRγ response elements (ERREs) within the KLK1 and KCNE1 5'-flanking regions required for ERRγ -stimulated transcriptional activity. Binding of endogenous ERRγ 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 ERRγ knockdown involved in cell differentiation, adhesion, and synthesis of steroid and peptide hormones required for placental development and function. Collectively, these findings suggest that ERRγ 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 ERRγ shRNA, or a control, nonsilencing shRNA. RNA from three independent experiments using placental tissues from three abortuses was extracted by miRNeasy® Mini Kit (Qiagen, Maryland, USA). RNA was labeled and hybridized to an Illumina HumanHT-12 v4 BeadChip, according to the manufacturer’s protocol.