Profiling hepatic microRNAs in zebrafish: Fluoxetine exposure mimics a fasting response that targets AMP-activated protein kinase (AMPK)
ABSTRACT: Recent evidence has suggested that fluoxetine, a serotonin-reuptake inhibitor and emerging environmental contaminant, can have non-targeted effects on metabolism in fish exposed to this waterborne pollutant. Using the highest, environmentally relevant, detectable level of fluoxetine (540 ng/L) we examined the impact of fluoxetine on the miRNA profile in the liver of zebrafish that were both fed and fasted for a period of 7 days. These results were further compared to the miRNA profile of zebrafish fasted and fed for 7 days, which were not exposed to fluoxetine. Results indicated that several miRNA that were involved with downregulating genes/pathways in response to fasting were also upregulated in fish exposed to fluoxetine, irrespective to fasting or feeding. These results suggest fluoxetine can have non-targeted effects on metabolic pathways mediated through miRNA expression. Furthermore, specific miRNA (dre-let-7d & dre-miR-140-5p) were found to target the catalytic subunit (AMPKa1 & AMPKa2, respectively) of AMP-Kinase, a master regulator of metabolism. Using predictive software and qPCR validation, combined with the expression profile of these two miRNA, we were able to establish a significant relationship between the expression of these specific miRNA to the downregulation of AMPKa subunit under the influence of 540 ng/L fluoxetine. Adult, female zebrafish were either fed or fasted for 7 days with and without the presense of 540 ng/L fluoxetine, and livers extracted and miRNA purified for miRNA microaary experiment.
Project description:Comparison of transcriptome data in fed and fasted conditions of zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss) anterior intestine using the serial analysis of gene expression (SAGE) method was done in a strategy to identify key regulated genes at the transcript level and to allow the identification of activated/deactivated pathways after feeding. Overall design: For the fasted group of zebrafish, total RNAs from the anterior intestines of a pool of 6 animals were used to generate the SAGE library (14bp TAGs, including the 4-bp anchor enzyme site) using the I-SAGE kit (Invitrogen, Cergy Pointoise, France). For the fed group of zebrafish (pool of 6 animals), and the fed and fasted trout (pool of 8 animals each), total RNAs extracted from the anterior intestine were used to generate long-SAGE libraries (20pb TAGs, including the 4-bp anchor enzyme site) using the I-SAGE Long kit (Invitrogen, Cergy Pointoise, France).
Project description:MicroRNAs (miRNAs) are short single-stranded RNA molecules that have a critical role in the regulation of gene expression. Alterations in miRNA expression levels have been observed in multiple tumor types and there is clear evidence on their active involvement in cancer development. Here, a comprehensive miRNA expression profiling in 16 pancreatic cancer cell lines and four normal pancreatic samples provided a specific molecular signature for pancreatic cancer and enabled us to identify 72 differentially expressed miRNAs with approximately half of them being up- and half downregulated in cancer cells as compared to normal samples. Of these, miR-31 was selected for further functional analyses based on its interesting “on-off” type expression profile, i.e. very low or even absent expression in normal pancreas and in six of the pancreatic cancer samples but extremely high expression in the remaining ten cell lines. Quite unexpectedly, both the inhibition of miR-31 in AsPC-1 and HPAF-II pancreatic cancer cells with high endogenous expression and forced expression of miR-31 in MIA PaCa-2 with low endogenous levels led to reduced cell proliferation, migration and invasion. More importantly, in AsPC-1 cells further enhancement of miR-31 also resulted in reduced cell migration and invasion, implicating that the level of miR-31 is critical for these phenotypes. We also identified novel miR-31 target genes, APBB2 and RSBN1, that might contribute to cancer pathogenesis. This study highlights a specific miRNA expression pattern in pancreatic cancer and reveals that manipulation of miR-31 expression leads to reduced cell migration and invasion in pancreatic cancer. 16 pancreatic cancer cell lines and 4 normal pancreatic RNA samples were hybridized on Agilent vs1 miRNA microarrays. Pool of four normal samples was hybridized on each slide to allow normalization between slides.
Project description:HTLV-1 infected individuals stay as carriers for their lifetimes, while the rest of 5% are killed by ATL. ATL leukemogenesis is a complex process involving accumulation of multiple genetic abnormalities in HTLV-1 infected cells. To clarify genetic events underlying ATL leukemogenesis, we conducted comprehensive miRNA expression profiling in 40 ATL patients and in 22 healthy volunteers. Total RNA samples from PBMC of ATL patients (mostly HTLV-1 inefcted CD4+ T-cells) and from CD4+ T-cells from healthy donors were subjected to Cy-3 labeling followed by miRNA expression microarray analyses.
Project description:Global microRNA expression profiling of microdissected pancreatic tissues were collected using Agilent miRNA microarrays (G4470B, Sanger 10.1) carrying 723 individual human miRNA probes. Four different sources of RNA were analyzed: microdissected normal pancreatic ductal cells (ND, n=4),microdissected acinar cells (AC, n=4), macrodissected chronic pancreatitis (CP, n=5) and micordissected xenograft tissues derived from primary pancreatic ductal adenocarcinomas (PDAC, n=5). Four condition (AZ, ND, PDAC, CP), each condition is represented by 4 to 5 biological replicates
Project description:MicroRNAs are a class of molecular regulators found to participate in numerous biological processes, including adipogenesis. However, whether dietary changes impact on microRNA (miRNA) in ruminants has not been reported. Therefore, this study aimed to evaluate the dietary effect on miRNA expression in subcutaneous (backfat) and visceral fat depots (perirenal fat) from beef steers fed with different diets containing high or low fat levels. Fat tissues were collected from 16 Hereford x Aberdeen Angus cross bred steers (15.5 month old) fed high fat diet (5.85% fat, n=8) or control diet (1.95% fat, n=8). Total RNA from each animal was subjected to miRNA microarray analysis using a customized Agilent miRNA microarray containing 672 bovine miRNA probes. Expression of miRNAs was not equally detected under two diets; 169 miRNAs were commonly expressed while 75 were diet specific. The number of miRNAs detected per animal under high fat diet was higher than those fed control diet (p= 0.037 in subcutaneous fat and p= 0.002 in visceral fat).. Further qRT-PCR analysis confirmed that the expression of some miRNAs was highly influenced by diet (miR-19a, -92a, -92b, -101, -103, -106, -142-5p, and 296) or fat depot (miR-196a and -2454). Our results revealed that the miRNA expression can be influenced by types of fat tissues or diet, suggesting that miRNAs may regulate bovine adipogenesis when diet alters. In this study, a total of 32 adipose tissue samples were analyzed by microRNA microarrays, being 16 subcutaneus (backfat) and 16 visceral (perirenal fat) fat depots were collected from 16 animals (Control diet = 8) (High fat diet = 8).
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:As microarray based gene expression profiling is well suited to study the complex diseases such as obesity, we revealed gene expression changes of fat tissues on obesity model zebrafish to elcidate the pathophysiological function of each fat tissue in metabolic syndrome. Zebrafish in over-feeding group were fed three times per day with Artemia (60 mg cysts/fish/day) through 8weeks. 1week over-feeding group were fed three times per day with Artemia (60 mg cysts/fish/day) through 1week. For caloric restriction, zebrafish were fed with Artemia (2.5 mg cysts/fish/day) for 2 weeks after over-fed with Artemia for 8 weeks.
Project description:102 postpubertal Holstein dairy heifers were randomly assigned to a fed or fasted treatment. Liver biopsies were performed to obtain tissue from which mRNA was extracted and used for microarray analysis. Transcriptional profiling revealed adaptive mechanisms in response to negative energy balance. Two condition experiment; fed and fasted. Common reference design employed; reference sample consisting of RNA derived from bovine liver, spleen and placental tissue. Biological replicates of 51 Fed (as control) and 51 Fasted (as treatment). One replicate per array. Dye swaps performed on 7 of the 51 fasted treatment group samples, and on 5 of the 51 fed treatment group samples.
Project description:Pharmaceutical chemicals used in human medicine are released into surface waters via municipal effluents and pose a risk for aquatic organisms. Among these substances are selective serotonin reuptake inhibitors (SSRIs) which can affect aquatic organisms at sub ppb concentrations. To better understand biochemical pathways influenced by SSRIs, evaluate changes in the transcriptome, and identify gene transcripts with potential for biomarkers of exposure to SSRIs; larval zebrafish Danio rerio were exposed (96 h) to two concentrations (25 and 250 µg/L) of the SSRIs, fluoxetine and sertraline, and changes in global gene expression were evaluated (Affymetrix GeneChip® Zebrafish Array). Significant changes in gene expression (>=1.7 fold change, p<0.05) were determined with Partek® Genomics Suite Gene Expression Data Analysis System and ontology analysis was conducted using Molecular Annotation System 3. The number of genes differentially expressed after fluoxetine exposure was 288 at 25 µg/L and 131 at 250 µg/L; and after sertraline exposure was 33 at 25 µg/L and 52 at 250 µg/L. Five genes were differentially regulated in all treatments relative to control, suggesting that both SSRIs share some similar molecular pathways. Among them, expression of the gene coding for FK506 binding protein 5 (FKBP5), which is annotated to stress response regulation, was highly down-regulated in all treatments (results confirmed by qRT-PCR). Gene ontology analysis indicated that regulation of stress response and cholinesterase activity were critical functions influenced by these SSRIs, and suggested that changes in the transcription of FKBP5 or acetylcholinesterase could be useful biomarkers of SSRIs exposure in wild fish. Zebrafish (Danio rerio) were obtained from the Zebrafish Research Facility maintained at the Center for Environmental Biotechnology at the University of Tennessee. Fish husbandry, spawning, and experimental procedures were conducted with approval from the UT Insititutional Animal Care and Use Committee (Protocol #1690-1007). Water for holding fish and conducting experiments (hereafter referred to as fish water) consisted of MilliQ water (Millipore, Bedford, MA) with ions added: 19 mg/L NaHCO3, 1 mg/L sea salt (Instant Ocean Synthetic Sea Salt, Mentor, OH), 10 mg/L CaSO4, 10 mg/L MgSO4, 2 mg/L KCl. Embroyos were obtained by spawning adult fish with no history of contaminant exposure. Fertilization of embryos took place at the same time (<15 minutes), such that larvae used in experiments were of similar age at the time of exposure. All activities (maintenance of adult fish, spawning, and experiments) were conducted in an environmental chamber with a temperature of 27 +/- 1 C and 14:10h light:dark photoperiod. Larval zebrafish (72 hpf) were exposed for 96 h in 200ml fish water containing appropirate amount of SSRI stock (i.e. fluoxetine or sertraline). There were four SSRIs treatments (25 and 250 ug/L fluoxetine and 25 and 250 ug/L sertraline) and one control (no SSRIs) with triplicate beakers and each beaker contained about 100 larval fish. During exposure for 96 hours, beakers were kept covered to prevent water evaporation and fish were not fed (i.e., fish consumed their yolk sac).