Project description:Here, we investigated the time-course changes in the pattern of microRNA (miRNA) expression of TNFα and IFNγ-stimulated and unstimulated hCMEC/D3 cells, an immortalized human cerebral microvascular endothelial cell line. In order to investigate pro-inflammatory cytokine-induced changes in miRNA levels in hCMEC/D3 cells, we challenged brain endothelial cells with TNFα and IFNγ (100 ng/ml) for 2 h, 6 h and 24 h and determined microRNA expression in cytokine-stimulated and unstimulated cells
Project description:The experiment was designed to look at the effect (on gene expression) of exposing human cerebromicrovascular endothelial cell line hCMEC/D3 to propionate (3 micromolar) for 24 h, to determine if circulating metabolites have the potential to affect integrity of the blood-brain barrier.
Project description:The experiment was designed to look at the effect (on gene expression) of exposing human cerebromicrovascular endothelial cell line hCMEC/D3 to trimethylamine (0.4 micromolar) or trimethylamine N-oxide (40 micromolar) for 24 h, to determine if circulating metabolites have the potential to affect integrity of the blood-brain barrier.
Project description:The molecular mechanisms playing a role in TWEAK versus TNFɑ signaling on cerebral microvascular endothelial cells are not well defined. Therefore, we aimed to identify gene expression changes in cultures of human brain microvascular endothelial cells (hCMEC/D3) to address changes initiated by TWEAK exposure. Although related, these two cytokines mediated different effects on gene expression and on canonical pathway activation. We used Agilent Whole Human Genome Microarray 4X44K to compare TWEAK- and TNFɑ-mediated changes in gene expression after 3h, 12h, and 24h incubation of these cytokines.
Project description:The study aimed to identify circular RNAs (circRNAs) commonly back-spliced to intronic region of different sets of endothelial cells (human cardiac microvascular endothelial cells (HCMEC), human aortic endothelial cells (HAoEC), human umbilical vein endothelial cells (HUVECs)) and to evaluate their overall expression and their expression compared to their respective host gene. Identified circRNAs were quality controlled by their detection in an additional exonuclease RNase R treated RNA-Seq dataset performed with RNA of HUVECs. CircRNAs were compared for overlapping detection between datasets and filtered by annotation for circRNAs back-spliced to intronic regions. Common endothelial intronic circRNAs candidates were compared to respective murine circRNAs stored in the circATLAS database. The prime candidate cZNF292 was functionally characterized in vivo and in vitro.
Project description:Transcriptional profiling of hCMEC/D3 endothelial cells after six hour incubation with conditioned media from unstimulated or LPS-stimulated monocyte derived macrophages
Project description:Over-expression of miR-155 induces changes in the pattern of gene expression of hCMEC/D3 cells. hypothesis tested in the present study was that miR-155 constitute an important regulatory control of the brain endothelial response to inflammatory cytokines. To identify miR-155 target genes in brain endothelim that might be implicated in BBB dysfunction relevant to human disease, we then analysed changes in mRNA expression of hCMEC/D3 cells that overexpress miR-155 and results were contrasted to cells transfected with scrambled miR. To ectopically express miR-155 in hCMEC/D3 cells, 30 nM of pre-miR-155 and the siPORT Amine transfection agent (Applied Biosystems, Warrington, UK) were combined following the manufacturerM-bM-^@M-^Ys instructions.
Project description:Functional and structural dysfunction of the blood brain barrier (BBB) leads to severe alterations in brain physiology and is believed to trigger neurodegeneration. To investigate the molecular mechanisms driving the BBB dysfunction, very few human BBB cell culture models are available; of which, the human microvascular endothelial cell line (hCMEC/D3) is the most widely used. Thus far, array-based approaches or targeted seqeuncing based approaches have been employed to characterize the gene expression of the hCMEC/D3 model. However,The goal of this study is to perform deep transcriptomic sequencing of the BBB cell line and obtain features like gene expression, expressed single nucleotide variants, alternate splice forms, circular RNAs, long non-coding RNAs and micro RNAs. We have developed blood brain barriers transcriptomics landscape using RNA sequencing and micro RNA seqeuncing data obtained from replicates of hCMEC/D3 BBB cell line.
Project description:Angiogenesis, the formation of new capillaries by sprouting from preexisting vessels, is mainly induced by VEGF-A. To identify genes which are induced by VEGF-A in endothelial cells, HUVEC were starved and induced by VEGF-A165 for 30, 60 and 150min. RNA of induced and uninduced cells was isolated and subjected to microarray analysis using Affymetrix microarray. Experiment Overall Design: Human umbilical vein endothelial cells were grown in complete EGM-2 medium in dense culture for four days without change of medium and before the stimulation in EGM-2 medium without growth factors over night. The cells were stimulated with 100ng/ml VEGF-A165 for 30, 60 and 150 min. RNA was isolated with Trizol according to the manufacturer's instructions.
Project description:To date, little evidence of miRNA expression/deregulation in multiple myeloma (MM) has been reported. To characterize miRNA expression profiling of MM plasma cells (PCs) and integrate miRNA expression data with other molecular features of MM patients, global miRNA expression profiles were generated for PCs isolated from BM biopsies of 38 newly diagnosed MM, 2 plasma cell leukemia patients, and 3 healthy donors; the samples were also profiled for global gene expression, and nineteen of them underwent genome-wide DNA analysis using high-density SNP-microarrays. Differential miRNA expression patterns were mainly identified in association with the major IGH translocations: in particular, t(4;14) showed specific over-expression of let-7e, miR-125a-5p and miR-99b belonging to a cluster at 19q13.33. The occurrence of other lesions, such as 1q gain, 13q and 17p deletions, and hyperdiploidy, was less well characterized by specific miRNA signatures. Genome-wide analysis showed that some allelic imbalances led to the significantly altered expression of miRNAs located in the involved regions, such as let-7b at 22q13.31 loss and miR-142-3p at 17q22 gain. The integrative analysis based on computational target prediction, miRNA and mRNA profiling defined a network of putative functional miRNA-target regulatory relations supported by expression data. This series of microarray experiments contains the microRNA profiles of purified plasma cells (PCs) obtained from 3 normal donor (N), 38 multiple myeloma (MM) and 2 plasma cell leukemia (PCL) at diagnosis. PCs were purified from bone marrow specimens after red blood cell lysis with 0.86% ammonium chloride using CD138 immunomagnetic microbeads. The purity of the positively selected PCs was assessed by morphology and flow cytometry and was >90% in all cases. 500 nanograms of total RNA was processed in accordance with the manufacturer's protocols (Agilent Technologies) to generate Cy3-labeled RNA which were purified on chromatography columns (Micro Biospin 6, Bio-Rad, Hercules, CA) and hybridized on an Agilent microarray (G4470B) at 55°C for 17 hr in a rotating oven. Images at 5 um resolution were generated using an Agilent scanner G2505B. The Feature Extraction 9.5 software (Agilent Technologies) was used to obtain the raw microarray data. The human miRNAs included in the platform were annotated according to Sanger miRBase Release 12.0. After discarding non-human miRNAs, the data were normalized using the Aroma Light package for Bioconductor. To overcome scaling biases due to background subtraction, the data were converted to obtain positive values throughout the dataset, at a minimum value of 1. The global gene expression raw data used in this study was originally deposited as GSE13591 and renormalized for this study. The genome wide profile (DNA) analysis was originally deposited as GSE16121.