Murine small intestinal crypt cells: IGF-1 stimulated vs. control
ABSTRACT: microRNA profiling of mouse small intestinal crypt cells comparing control untreated with cells treated with insulin growth factor-1 (IGF-1). IGF-1 stimulated cell proliferation, as observed in Brdu incoporation assay. Two condition experiment. Control vs IGF-1 treatment. Biological replicates: 3 control, 3 treated. Independently grown and harvested. One replicate per array
Project description:microRNA profiling of rat small intestinal crypt cell IEC-6. Comparing control untreated with cells treated with transforming growth factor-beta (TGF-beta). TGF-beta stimulated cell differentiation, as observed in the stimulation of intestinal epithelial cell markers (alkaline phophotase, villin, aminopeptidase N, etc.). Two condition experiment. Control vs TGF-beta treatment. Biological replicates: 3 control, 3 treated. Independently grown and harvested. One replicate per array
Project description:Comparison of non-coding RNA profiling by array in sublines of DU145 human prostate cancer cell lines created by in vivo cycling Cell lines created by removal and growth of metastatic human DU145 tumor cells from mouse lymph node (metastasized from prostate xenograft) for LN cells and extracted from lung after intravenous injection (ivLU cells). Cell line numebr represented number of in vivo cycles of metastatic selection Six condition experiment, individual cell line for each sample on array
Project description:We report the identification of microRNA-138 (miR-138), as a molecular signature of GSCs and demonstrate a vital role for miR-138 to promote growth and survival of bona fide tumor-initiating cells with self-renewal potential. Total RNA from Glioma Stem Cells and Neural Stem Cells were subjected to microRNA microarray analysis, 3 replicates each.
Project description:Pterygium is a relatively common human ocular surface fibroproliferative disease that affects vision. Endogenously produced microRNA (miRNA) regulates gene expression in various ocular surface diseases and possibly pterygium. We aimed to investigate the role of miRNA in pterygium. Paired human pterygium and conjunctival tissues were obtained from patients diagnosed with primary pterygium. miRNA microarray profiling identified statistically significant miRNA changes which were matched to reciprocal significant changes in their target transcripts. We employed quantitative real-time polymerase chain reaction and found that hsa-miR-766 was up-regulated (2.57-fold) whilst hsa-miR-215 was down-regulated (0.49-fold) in pterygium compared to conjunctival control. Localization of miRNA was performed using in-situ hybridization. Transcript levels of predicted hsa-miR-766 targets, nuclear receptor subfamily 4, group A, member 1 and epidermal growth factor-containing fibulin-like extracellular matrix protein 1, were down-regulated in pterygium compared to conjunctiva by 0.53- and 0.64-fold, respectively. Collagens type 3, alpha 1 and type 4, alpha 2, both targets of hsa-miR-215, were up-regulated in pterygium by 3.01- and 3.11-fold, respectively. These changes were confirmed in the protein levels using immunofluorescent staining. Derangement of hsa-miR-766 and hsa-miR-215 may cause dysregulation of matrix rearrangement, cell proliferation and adhesion proteins, resulting in pterygium formation. Targeting miRNA may be a possible therapeutic approach in this disease. 3 pterygium samples and 3 matched conjuctiva samples from patients diagnosed with primary pterygium. A pool of all 6 samples was used as the common reference.
Project description:Lack of change in microRNA expression in adult mouse liver following treatment with benzo(a)pyrene (BaP), as detected using Exiqon miRNA arrays. Adult male mice were exposed to 150 mg/kg benzo(a)pyrene (BaP) or solvent for 3 days and sampled 4 hours after the last dose. MicroRNA expression levels in adult mouse liver were measured using Exiqon miRNA arrays. Our results indicate a distinct lack of effect of BaP of miRNA expression, despite widespread changes in mRNA levels (measured using Agilent arrays). Lack of miRNA changes was confirmed with Agilent miRNA arrays. Keywords: Toxicology, miRNA Adult male B6C3F1 mice were exposed to a daily dose of corn oil (vehicle control group) or 150 mg/kg BaP (treatment group) for 3 d (n=6 per group). Mice were sacrificed at 4 h or 24 h following the last dose and liver lobes were extracted and flash frozen. Exiqon miRNA arrays were used to examine changes in miRNA transcript levels in random liver lobe sections.
Project description:Using microarray, we compared the expression of miRNAs from the peripheral blood of male subjects with T1DM and T2DM with healthy controls. Healthy male controls used were age-matched to the T2DM group patients with mean(SD) 37.3 (7.1) years. Subjects with T1DM were younger [23.3(1.6) years]. Expression was compared and validated using quantitative real-time PCR. Statistical testing (ANOVA, P-value <0.05) was performed and fold changes with respect to the control were calculated Systolic BP, fasting glucose, HbA1c, total cholesterol and LDL-cholesterol levels were significantly higher in T2DM subjects compared with controls (P-value <0.05). Compared with controls, we identified 37 differentially regulated miRNAs in DM subjects. Among them, 21 miRNAs were upregulated (2-5 fold change, p-value < 0.05) and 16 miRNAs were downregulated (1.5-2 fold change, p-value < 0.05). These miRNAs had gene putative targets primarily involved in regulating pancreatic development and functions, adipocyte differentiation, insulin signaling and glucose-dependent insulin secretion. peripheral blood of male subjects with T1DM and T2DM with healthy controls
Project description:Dysregulation in expression of microRNAs (miRNAs) in various tissues has been linked to a wide spectrum of diseases, including Type 2 Diabetes mellitus (T2D). In this study, we compared the expression profiles of miRNAs in blood samples from Impaired Fasting Glucose (IFG) and T2D male patients with tissues from T2D rat models. Healthy adult males with no past history of T2D (n=158) and with desirable cholesterol and blood pressure profiles were enrolled in this study. They were then classified according to fasting glucose levels to have T2D, IFG or as healthy controls (CTL), for comparison of miRNA expression profiles. Employing miRNA microarray, we identified ‘signature miRNAs’ in peripheral blood samples that distinguished IFG and T2D. Eight selected miRNAs were further validated using stem-loop real-time RT-PCR. miR-144 expression was found to be dysregulated in Type 2 Diabetes, wherein its expression was significantly higher than in healthy controls. Insulin receptor substrate 1 (IRS1) has been predicted to be a potential target of miR-144. Consistent with this observation, IRS1 mRNA and protein levels, verified by quantitative real-time PCR and western blotting respectively, were found to be down-regulated. Using luciferase assay, we further demonstrated that miR-144 directly targets IRS1 and showed its effects on protein expression via immunocytochemistry. From this cross-sectional study in humans, we have identified signature miRNAs which could explain the pathogenesis of T2D. Whether miRNAs like miR-144 could be potential therapeutic targets for management of T2D will need to be explored by further mechanistic and functional studies. miRNA profiling of tissues from T2D rat models. Total RNA (plus miRNAs) was isolated using a modification of the RiboPure™-Blood kit from Ambion (Austin,TX) according to the manufacturer’s protocol. The concentration of total RNA and integrity were determined by using Nano-Drop ND-1000 Spectrophotometry (NanoDrop Tech, Rockland, Del) and gel electrophoresis respectively.
Project description:Brain tumor neurospheres (BTCSs) are cancer cells with neural stem cell-like properties found in the fatal brain tumor glioblastoma multiforme (GBM). These cells account for less than 1% of total tumor cells, are poorly differentiated and are believed to be involved in tumor induction, progression, treatment resistance and relapse. Specific miRNAs play important roles in modulating the proliferation and differentiation of neural stem cells, therefore, we aimed to identify miRNAs controlling differentiation in GBM-BTSCs through high throughput screening miRNA array profiling. We compared the miRNA expression profiles at the neurosphere state and upon 4 and 14 days of differentiation by using LIMMA, finding 21 differentially expressed miRNAs : hsa-miR-103, hsa-miR-106a, hsa-miR-106b, hsa-miR-15b, hsa-miR-17, hsa-miR-19a, hsa-miR-20a, hsa-miR-25, hsa-miR-301a and hsa-miR-93 were found up-regulated upon differentiation, while hsa-miR-100, hsa-miR-1259, hsa-miR-21, hsa-miR-22, hsa-miR-221, hsa-miR-222, hsa-miR-23b, hsa-miR-27a, hsa-miR-27b, hsa-miR-29a and hsa-miR-29b were down-regulated. Expression of 11 of the 21 miRNAs was examined by qPCR and 7 of them were validated: hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222 increased their expression upon differentiation, while hsa-miR-93 and hsa-miR-106a were inhibited. Functional studies demonstrated that miR-21 over-expression induced the expression of glial and/or neuronal cell markers in the neurospheres, possibly due to SPRY1 targeting by miR-21 in these cells, while miR-221 and miR-222 inhibition at the differentiated state reduced the expression of those differentiation markers. On the other hand, miR-29a and miR-29b targeted MCL1 in the GBM neurospheres and increased apoptotic cell death. Five GBM cell lines at the neurosphere state or after 4 or 14 days of differentiation
Project description:Invariant Natural killer T (iNKT) cells are a separate lineage of T lymphocytes with innate effector functions. They express an invariant TCR specific for lipids presented by CD1d and their development and effector differentiation rely on a unique gene expression program. We asked whether this program includes microRNAs, small non-coding RNAs that regulate gene expression posttranscriptionally and play key role in the control of cellular differentiation programs. We identified a miRNA profile specific for iNKT cells, which exhibits features of activated/effector T lymphocytes. In this experiment we compared microRNAs of NKT cells versus those of conventional T lymphocytes, both extracted from wild type mouse thymus. To produce the populations we enriched mature thymocytes and then sorted NKT cells and conventional T cells (from one sorting we obtain one “NKT” sample and one “T” sample). We performed the experiment in triplicate; the sample NKT 1 was sorted the same day of the sample T 1, NKT 2 with T 2, NKT 3 with T 3. We used a common reference approach for the 6 samples; as a common reference we produced RNA from total thymocytes, without the enrichment for mature cells and without the sorting; we pooled thymocytes derived from all the thymi used in the study. The aim of the experiment was to demonstrate that NKT cells have a microRNA profile different from that of conventional T cells.
Project description:Amnion is a vital structure for human parturition, and it is anatomically compartmentalized into the placental amnion and the reflected amnion. microRNA expression was profiled to determine the expression pattern and its significance in the placental amnion and the reflected amnion in association with spontaneous labor at term. placental and reflected amnion tissue from two groups of women (term in labor and term no labor)