Project description:Transcriptional profiling of miRNA levels in mammary tumors from 18 [PyMT x AKXD]F1 sublines. The PyMT strain was FVB/N-TgN(MMTV-PyVT)634Mul. Mammary tumor total RNA from mice representing one of 18 AKXD RI strains were pooled to represent each strain and expression profiled using a custom miRNA microarray.
Project description:Total RNA was extracted from adipose tissue of high (full) fat diet and standard fat diet mice. Adipose tissue was taken from 16 mice in total. Eight mice were fed a standard diet (SD; control) (10 kcal% fat) and the other eight were fed a high-fat diet (HFD; test) (60 kcal% fat; Research Diets, New Brunswick, NJ) for 5 months. Total RNA was isolated from pooled White Adipose Tissue from SD- or HFD-fed mice using guanidinium thiocyanate. The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile. One µg total RNA from sample and reference were labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA Array power labeling kit (Exiqon, Denmark) following the procedure described by the manufacturer. The Hy3™-labeled samples and the Hy5™-labeled sample were mixed pair-wise and hybridized to the miRCURY™ LNA array version 10.0 (Exiqon, Denmark), which contains capture probes targeting all miRNAs for all species registered in the miRBASE version 11.0 at the Sanger Institute. The hybridization was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station (Tecan, Austria). After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY™ LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene 8.0 software (BioDiscovery, Inc., USA). The quantified signals were normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm.
Project description:Profiling of circulating miRNAs before, 60 mins, 1day and 3 days after an acute resistance exercise. Three subjects performed an acute resistance exercise. The resistance exercise consisted of two consecutive exercises (bench press and bilateral leg press), consisting of five sets of 10 repetitions at 70% of 1 RM. The subjects were allowed to rest for 1 min between each set and exercise. The subjects were instructed to lift and lower the load at a constant velocity, taking about 2 s for each repetition. If the load became too heavy, the subject was assisted. The range of motion in each exercise was from 90° to 0° (0° at full extension). Blood samples were taken before, 60 mins, 1day and 3days after the exercise.
Project description:Cardiomyocytes derived from human pluripotent stem cells were exposed to the cardiotoxic drug Doxorubicin in order to assess the utility of this cell system as a model for drug-induced cardiotoxicity. Cells are exposed to different concentrations of doxorubicin for up to 48 hours followed by a 12 days recovery period.
Project description:The human pathogen Helicobacter pylori (Hp) colonizes the gastric epithelium as a unique niche in the stomach. Infections commonly occur in childhood and persist lifelong, leading in some cases to adenocarcinoma and MALT lymphoma. Several studies define mammalian microRNAs as key regulators of the immune system and of carcinogenesis processes. Here, we show Hp infection induces miR-155 expression in epithelial and hematopoietic cells in vitro and in vivo. This induction is mediated by at least two main bacterial virulence factors, the vacuolating toxin (VacA) and the gamma-glutamyl transpeptidase (GGT) in an LPS independent manner. Using adenylate cyclases agonists and inhibitor, we demonstrated that the miR-155 microRNA regulatory cascade in Hp infection involves the second messenger cyclic adenosine monophosphate (cAMP). Furthermore, the study showed that a knock-down of the Foxp3 transcription factor in T cells abolishes miR-155 expression. Together, these findings are the first demonstration of a direct interconnection between the regulatory T cell development factor Foxp3 and a microRNA. This study supports the link between Hp infection, regulation of cellular immunity, inflammation and cancer development. A color-swap dye reversal experimental setting was applied. Ratio profiles comprising single hybridizations were combined in an error-weighted fashion to create ratio experiments. A two-fold change expression cut off for ratio experiments was applied together with anti-correlation of ratio profiles rendering the microarray analysis set highly significant (P-value > 0.01), robust and reproducible.
Project description:Both disseminated tumor cells and noncancerous host cells contributed to cancer progression cooperatively in bone marrow. Bone marrow samples were obtained from 4 gastric cancer patients, and were separated into 3 fractions (CD45 positive, CD45 negative/EpCAM positive, and CD14 positive fractions) by the automagnetic-activated cell separation (AutoMACS) system using CD45, EpCAM, and CD14 microbeads (Miltenyi Biotec, Germany). microRNA expression profiles in each fractions were evaluated in order to identify candidate prognostic markers for gastric cancer patients. In 4 patients with gastric cancer, bone marrow samples (40 mL) were obtained from iliac bones. Nucleated cells were collected by gradient centrifugation using Ficoll-Paque PREMIUM (GE Healthcare Life Science, USA) and Leucosep (Greiner Bio-One, Germany) according to the manufacturer’s instructions. Next, we separated bone marrow cells into 3 fractions using MACS: CD45 positive (CD45+), CD45 negative/EpCAM positive (CD45-/EpCAM+), and CD14 positive (CD14+). microRNA expression levels of whole bone marrow cells and each fractions were measured by the miRCURY™ LNA array microarray (6th gen-hsa, mmu & rno#208402, Exiqon, Vedbaek, Denmark). The miRCURY™ LNA array microarray slides were scanned using the Agilent G2505C Microarray Scanner System (Agilent Technologies, Inc., USA) and the data analysis was carried out using the Feature Extraction 10.7.3.1 (Agilent Technologies, Inc., USA).
Project description:To better understand the molecular mechanisms of SP cells, we screened the miRNAs expression patterns in the SP compared with the MP cells. MiRCURY⢠LNA array analysis of sorted SP and MP cells from two relapsed myeloma patients with more than 70% bone marrow plasma cells were performed. In order to identify the miRNA patten between SP and MP cells, the samples were labeled using the miRCURY⢠Hy3â¢/Hy5⢠Power labeling kit (Exiqon) and hybridized on the miRCURY⢠LNA Array (v.16.0).
Project description:Over 40 % of microRNAs are located in introns of coding genes, and many intronic microRNAs are co-regulated with their host genes. In such cases of co-regulation, the products of host genes and their intronic microRNAs can cooperate to coordinately regulate biologically important pathways. Therefore, we screened intronic microRNAs dysregulated in liver of obese mouse models to identify previously uncharacterized coding host genes that may contribute to the pathogenesis of obesity-associated insulin resistance and type 2 diabetes mellitus. Our approach identified that expression of both Ectodysplasin A (Eda), the causal gene of X-linked hypohidrotic ectodermal dysplasia (XLHED; MIM 305100) and its intronic microRNA, miR-676, was strongly increased in liver of obese mouse models. Moreover, hepatic EDA expression is increased in obese human subjects, reduced upon weight loss, and its hepatic expression correlates with systemic insulin resistance. Eda expression in murine liver is controlled via PPARg activation, increases in circulation and promotes JNK activation and inhibitory serine phosphorylation of IRS1 in skeletal muscle. Consistently, bi-directional modulation of hepatic Eda expression in mouse models affects systemic glucose metabolism with alterations of muscle insulin signaling, revealing a novel role of EDA as an obesity-associated hepatokine, which impairs insulin sensitivity in skeletal muscle.
Project description:microRNAs were profiled in healthy controls, classic celiac patients (CD), CD patients with anemia and GFD treated CD with normalization of duodenal mucosa all CD conditions were related to controls. For each group, five patients were pooled. One replicate per experiment
Project description:mRNA expression data from mammary tumors extracted 30 days after orthotopic injection of miR-290-expressing and negative control 6dt1 cells into female FVB/N mice. At 6 weeks of age, FVB/N female mice were orthotopically injected with 1.0x10E5 6dt1 cells, either expressing miR-290 or a negative control RNA construct. 4 negative control tumors and 3 miR-290 tumors were analyzed.