MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported. Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146 overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs. The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Overall design: Cells were transfected with a plasmid to direct overexpression of miR-146a. Extracellular vesicles were isolated by ultracentrifugation from untreated and transfected cells. RNA was isolated from one sample each of untreated and transfected cells and vesicles.Small RNA libraries were prepared for sequencing.
Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types.
Specimen part, Subject
View SamplesThe inner ear develops from a patch of thickened cranial ectoderm adjacent to the hindbrain called the otic placode. Studies in a number of vertebrate species suggest that the initial steps in induction of the otic placode are regulated by members of the Fibroblast Growth Factor (FGF) family, and that inhibition of FGF signaling can prevent otic placode formation. To better understand the genetic pathways activated by FGF signaling during otic placode induction, we performed microarray experiments to estimate the proportion of chicken otic placode genes that can be up-regulated by the FGF pathway in a simple culture model of otic placode induction. Surprisingly, we find that FGF is only sufficient to induce about 15% of chick otic placode-specific genes in our experimental system. However, pharmacological blockade of the FGF pathway in cultured chick embryos showed that although FGF signaling was not sufficient to induce the majority of otic placode-specific genes, it was still necessary for their expression in vivo. These inhibitor experiments further suggest that the early steps in otic placode induction regulated by FGF signaling occur through the MAP kinase pathway. Although our work suggests that FGF signaling is necessary for otic placode induction, it demonstrates that other unidentified signaling pathways are required to co-operate with FGF signaling to induce the full otic placode program.
Analysis of FGF-dependent and FGF-independent pathways in otic placode induction.
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Tribbles 3: a novel regulator of TLR2-mediated signaling in response to Helicobacter pylori lipopolysaccharide.
Specimen part, Cell line
View SamplesThis study set out to identify global changes in gene expression in human embryonic kidney (HEK) cells stably transfected with Toll-like receptor 2 (TLR2) over a 48 hour time-course, following stimulation with 10 g/ml lipopolysaccharide (LPS) from the gastric pathogen H. pylori.
Tribbles 3: a novel regulator of TLR2-mediated signaling in response to Helicobacter pylori lipopolysaccharide.
Specimen part
View SamplesTo define H3K27me3 modified genes in intestinal stem, progenitor and epithelial cells, we performed chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq). We used RNA-sequencing to profile gene expression changes during intestinal stem cell differentiation into mature villus cells, as well as genes perturbed upon loss of PRC2 activity (deletion of Eed) . We find thousands of genes that change in expression including many H3K27me3 marked genes. The deregulated genes reaveal a intestinal tissue specific role of PRC2. Overall design: H3K27me3, H3K4me2 and RNA Pol2 ChIP-Seq analysis of isolated mouse intestinal stem cells, enterocyte and secretory progenitor cells, and mature villus cells. RNA seq analysis of control mouse villus cells, control intestinal stem cells and Eed-deleted villus.
Acquired Tissue-Specific Promoter Bivalency Is a Basis for PRC2 Necessity in Adult Cells.
No sample metadata fields
View SamplesThis study set out to identify global changes in gene expression in AGS gastric epithelial cells following 8 hours stimulation with 10 g/ml lipopolysaccharide (LPS) from the gastric pathogen H. pylori.
Tribbles 3: a novel regulator of TLR2-mediated signaling in response to Helicobacter pylori lipopolysaccharide.
Specimen part, Cell line
View SamplesThis study set out to identify global changes in gene expression in MKN45 gastric epithelial cells following 8 hours stimulation with 10 g/ml lipopolysaccharide (LPS) from the gastric pathogen H. pylori.
Tribbles 3: a novel regulator of TLR2-mediated signaling in response to Helicobacter pylori lipopolysaccharide.
Specimen part, Cell line
View SamplesThe aim of this study was to identify and quantify microRNAs and other small regulatory RNAs expressed in primary retinal microvascular endothelial cells (RMECs) using deep sequencing. RMECs were isolated, RNA extracted, a small RNA library prepared and deep sequencing performed. A total of 6.8 million reads were mapped to 250 known microRNAs in miRBase (release 16). Several novel microRNAs and multiple new members of the miR-2284/2285 family were detected. Several ~30 nucleotide sno-miRNAs were identified, with the most highly expressed being derived from snoRNA U78. Highly expressed microRNAs previously associated with endothelial cells included miR-126 and miR-378, but the most highly expressed was miR-21, comprising more than one third of all mapped reads. The independence from prior sequence knowledge provided by deep sequencing facilitates analysis of novel microRNAs and other small RNAs. This approach also enables quantitative evaluation of microRNA expression, which has highlighted the predominance of a small number of microRNAs in RMECs. Further characterisation of the functions of the highly expressed microRNAs will provide insights into endothelial biology. Overall design: Single sample of primary cell culture
Deep sequencing reveals predominant expression of miR-21 amongst the small non-coding RNAs in retinal microvascular endothelial cells.
Specimen part, Subject
View SamplesRegulation of gene expression is an important aspect of insulin's physiological action, however, most studies rely on in vitro systems or pharmacological doses of insulin. Here, we demonstrate that under euglycemic-clamp conditions, physiological levels of insulin regulate over 1500 transcripts in muscle and 1000 transcripts in liver. These include expected pathways related to glucose and lipid utilization, mitochondrial function and autophagy in muscle, and glucose production and steroidogenesis in liver, as well as unexpected pathways, such as mRNA splicing, chromatin remodeling, and regulation of hepatocyte nuclear factors. Insulin also regulates over 100 non-coding RNAs in muscle and liver. These changes in coding and non-coding RNAs, refined by alternative splicing, provide an integrated transcriptional network underlying the complexity of insulin action in vivo.
Multi-dimensional Transcriptional Remodeling by Physiological Insulin In Vivo.
Sex, Age, Specimen part, Cell line, Time
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Histone methyltransferase MLL3 contributes to genome-scale circadian transcription.
Specimen part, Time
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