Expression profiling of microRNAs in Bovine ovarian follicular development
ABSTRACT: The study aimed to identify miRNAs expression profiles associated with growth and regression of dominant-size follicles in bovine. Follicles were collected from abattoir ovaries and their status (healthy/atretic) was assessed by measuring steroid levels and aromatase expression. Total RNA was isolated from whole follicles at different developmental stages. An heterologous microarray (Exiqon, Denmark) approach followed by RT-qPCR validation (Qiagen, UK) was used to identify and compare miRNA profiles between large healthy follicles (diameter, 13–16 mm, n=6) and each of small (4–8 mm, n=6 pools of follicles) and large atretic folllicles (13-16 mm, n=6). RNA from the above groups was hybridized to the miRCURY LNA™ microRNA Hi-Power Labeling Kit,Hy3™/Hy5™ (Exiqon) and hybridized on the miRCURY LNA™ microRNA Array (6th gen). A total of 17 and 57 microRNAs were differentially expressed (> 2 fold, adj. P-value < 0.05) between Large Healthy and each of Small and Large Atretic follicles, respectively, a fraction of which corresponded to registered bovine miRNA sequences. A subset of 5 bovine miRNAs (miR-144, miR-202,vmiR-451, miR-652, miR-873) were confirmed by qPCR to be upregulated in Large Healthy follicles, were enriched in mural granulosa cells and their predicted targets mapped to genes involved in follicular cell proliferation and differentiation, suggesting an involvemet of this subset of microRNAs in ovarian follicle development. Six biological replicates per developmental stage (total of 18 samples) were used in a double dye microRNA microarray experiment. Samples were distributed among slides so that each experimental group was represented at least once in each slide. For each gene, mean normalized intensities (n= 6 biological replicates/group) were compared between follicle stages (SF vs LHF and LHF vs LAF).
Project description:Follicular dendritic cells (FDC) are important stromal cells within the B cell follicles and germinal centres (GC) of secondary lymphoid tissues. FDC trap and retain native antigens on their surfaces in the form of immune complexes which they display to B cells, in order to select those cells with the highest antigen affinity. MicroRNAs are short, non-coding RNAs of approximately 18-25 nucleotides in length that regulate gene expression at the post-transcriptional level by repressing the translation of target genes. In the current study in vivo and in vitro systems were used to identify microRNAs that are differentially expressed as a result of FDC depletion. Constitutive lymphotoxin-β receptor (LTβR) stimulation is required to maintain FDC in their differentiated state. We show that the rapid de-differentiation of spleen FDC that followed LTβR-blockade, coincided with a significant decrease in the expression of mmu-miR-100-5p, mmu-miR-138-5p and mmu-miR-2137. These microRNAs were shown to be expressed in the FDC-like cell line, FL-YB, and specific inhibition of mmu-miR-100-5p significantly enhanced expression of Il6, Ptgs1/2 and Tlr4 in this cell line. The expression of each of these genes by FDC plays an important role in regulating GC size and promoting high-affinity antibody responses, suggesting that mmu-miR-100-5p may help regulate their expression during GC reactions. C57BL/6 mice were given a single intravenous injection of 100 µg of LTβR to temporarily deplete their FDC. At intervals after treatment 4 spleens from each group were harvested and RNA prepared. For each group samples were pooled into 2 groups of 2 and microRNA expression levels compared. Spleens from LTb-/- mice were also analysed. One channel was used for the actual sample, the second channel was used for internal QC reference.
Project description:Results Platelets in non-diabetic patients demonstrated miRNA expression profiles comparable to previously published data. The miRNA expression profiles of platelets in diabetics were similar. Statistical analysis unveiled only three miRNAs (miR-377-5p, miR-628-3p, miR-3137) with high reselection probabilities in resampling techniques, corresponding to signatures with only modest discriminatory performance. Functional annotation of predicted targets for these miRNAs pointed towards an influence of diabetes mellitus on mRNA processing. Conclusions/interpretation We did not find any major differences in platelet miRNA profiles between diabetics and non-diabetics. Minor differences pertained to miRNAs associated with mRNA processing. Thus, previously described differences in plasma miRNAs between diabetic and nondiabetic patients cannot be explained by plain changes in the platelet miRNA profile. Platelet miRNA profiles were assessed in clinically stable diabetic and nondiabetic patients (each n=30). Platelet miRNA was isolated from leucocyte-depleted platelet-rich plasma, and miRNA profiling was performed using LNA micro-array technology (miRBase 18.0, containing 1,917 human miRNAs). Effects of diabetes mellitus were explored by univariate statistical tests for each miRNA, adjusted for potential confounders, and by developing a multivariable signature, which was evaluated by resampling techniques. Platelet phenotype was assessed by light transmission aggregometry and impedance aggregometry.
Project description:Background: T-cell intracellular antigen (TIA) proteins function as regulators of cell homeostasis. These proteins control gene expression globally at multiple levels in response to dynamic regulatory changes and environmental stresses. Herein we identified a micro(mi)RNA signature associated to transiently TIA-depleted HeLa cells and analyzed the potential role of miRNAs combining genome-wide analysis data on mRNA and miRNA profiles. Results: Using high-throughput miRNA expression profiling, transient depletion of TIA-proteins in HeLa cells was observed to promote significant and reproducible changes (>2-fold, FDR<0.0001) affecting to a pool of up-regulated miRNAs (miR-30b*, miR125a-3p, miR-193a-5p, miR-197_MM2, miR-203, miR-210, miR-371-5p, miR-373*, miR-483-5p, miR-492, miR-498, miR-503, miR-572, miR-586, miR-612, miR-615, miR-623, miR-625, miR-629, miR-638, miR-658, miR-663, miR-671, miR-769-3p and miR-744). Differential expression analysis of some miRNAs was validated by reverse transcription and real time PCR. By target prediction and combined analysis of the genome-wide expression profiles of the mRNAs and miRNAs identified in TIA-depleted HeLa cells, we detected concomitant connections between up-regulated miRNAs and putative and experimental targeted mRNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes database analyses suggest that targeted mRNAs are related with biological processes associated to the regulation of DNA-dependent transcription, signal transduction and multicellular organismal development as well as with the enrichment of pathways in cancer, focal adhesion, regulation of actin cytoskeleton and MAPK and Wnt signalling pathways, respectively. Conclusion: All this considered, these observations suggest that specific miRNAs could act as potential mediators of the epigenetic switch linking transcriptomic dynamics and cell phenotypes mediated by TIA proteins. The analysis includes two cell types. Three biological replicates were performed per cell type and they were compared by using three dual-channel microarray hybridizations.
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:Expression profiles of microRNAs in neonatal (isolated from day0 newborn rats) and adult rat cardiomyocytes (isolated from 2month old rats) Two condition experiment; Biological replicates: 7 samples of cardiomyocytes from neonatal rats (from independent isolations); 6 samples of cardiomyocytes isolated from adult animals (from independent isolations)
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: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:To determine if treatment with a combined therapeutic regimen, which resulted in clinical benefit in a subset of patients in a clinical trial, alters miRNA expression in a way that may be used to guide treatment with these agents and to identify miRNAs that may be involved in the mechanism of action of this regimen Three-condition experiment, untreated tissue, post-temsirolimus alone treatment, post combination-treatment tissue. 33 total samples, including 5 samples resubmitted for quality control.
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.