Project description:Analysis of the dentate gyrus of amygdala electrical stimulation model of temporal lobe epilepsy. Results provide insight into the molecular mechanism underlying epileptogenesis. This study was designed to estimate changes in miRNA expression levels after 7, 14, 30 and 90 days after electrical stimulation of amygdala as a model of temporal lobe epilepsy. The advantage of this study is time matched control (sham operated animals sacrificed at the same age as stimulated animals).
Project description:Sprouty proteins are evolutionarily conserved modulators of mitogen-activated protein kinase pathway. Sprouty2 appears to function as a tumor suppressor in cancers, whereas we reported earlier that Sprouty2 functions as an oncogene in colorectal cancer. To further understand the oncogenic potential of Sprouty2 in the colon, microRNA expression profile of colon cancer cells was investigated. Sprouty2 suppression in HCT116 colon cancer cells significantly increased MicroRNA 194-5p. Sprouty2 dependent regulation of microRNA194-5p and its biological targets were studied further for their tumor suppressive actions in reducing epithelial-mesenchymal transition in colorectal cancer. Sprouty2 knockdown was performed by infecting HCT116 cells with three different lentivirus expressing shRNAs against human Sprouty2 mRNA and a control non targeted non-silencing shRNA (Sprouty2 MISSION shRNA Lentiviral Transduction Particles; TRCN 0000007522, TRCN 0000231589, TRCN 0000231588 and a non-targeted shRNA control from Sigma) following lentiviral transduction protocols provided by Sigma. Due to the random integration of the lentivirus into the host genome, varying levels of Sprouty2 gene knockdown was expected in puromycin resistant colonies. Three colonies in triplicate that demonstrated highest to lowest level of Sprouty2 suppression, as assessed by western blotting, were selected. RNA samples from these colonies and one from non-targeted shRNA expressing colony were prepared for microRNA expression profile analysis. Pooled RNA samples from each group were shipped to Exiqon for microRNA profiling based on miRCURY LNATM array technology.
Project description:miRNAs were extracted from plasma samples of healthy volunteers, patients with Barrettâ??s esophagus and patients with esophageal adenocarcinoma. We used the Serum / Plasma Focus miRNA PCR panel (Exiqon, Denmark) to quantitate miRNA presence in the different patient groups. PCR miRNA profiling. miRNA from three different patient groups were extracted from their plasma.
Project description: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, 13M-bM-^@M-^S16 mm, n=6) and each of small (4M-bM-^@M-^S8 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 LNAM-bM-^DM-" microRNA Hi-Power Labeling Kit,Hy3M-bM-^DM-"/Hy5M-bM-^DM-" (Exiqon) and hybridized on the miRCURY LNAM-bM-^DM-" 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: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: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: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:Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders. Human fetal neurons were chosen to examine the impact of HIV-1 Vpr protein on gene expression
Project description:Over the last decade, small noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators in the expression and function of eukaryotic genomes. It has been suggested that viral infections and neurological disease outcome may also be shaped by the influence of small RNAs. This has prompted us to suggest that HIV infection alters the endogenous miRNA expression patterns, thereby contributing to neuronal deregulation and AIDS dementia. Therefore, using primary cultures and neuronal cell lines, we examined the impact of a viral protein (HIV-1 Tat) on the expression of miRNAs due to its characteristic features such as release from the infected cells and taken up by noninfected cells. Using microRNA array assay, we demonstrated that Tat deregulates the levels of several miRNAs. Interestingly, miR-34a was among the most highly induced miRNAs in Tat-treated neurons. Tat also decreases the levels of miR-34a target genes such as CREB protein as shown by real time PCR. The effect of Tat was neutralized in the presence of anti-miR-34a. Using in situ hybridization assay, we found that the levels of miR-34a increase in Tat transgenic mice when compared with the parental mice. Therefore, we conclude that deregulation of neuronal functions by HIV-1 Tat protein is miRNA-dependent. Human fetal neurons were chosen to examine the impact of HIV-1 Tat protein on gene expression
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-M-NM-2 receptor (LTM-NM-2R) stimulation is required to maintain FDC in their differentiated state. We show that the rapid de-differentiation of spleen FDC that followed LTM-NM-2R-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 M-BM-5g of LTM-NM-2R 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.