ABSTRACT: Reactive Oxygen Species (ROS) could be a stress factor that affects microRNA regulation and function in macrophages. The production of microRNAs (miRNA) is influenced by various stimuli, including environmental stresses. We hypothesized that ROS-associated stress could regulate macrophage miRNA synthesis. p47phox-/- mice have deficient NADPH oxidase activity resulting in decreased ROS production. We cultured bone marrow-derived macrophages (BMDM) from wild type (WT) and p47phox-/- mice and profiled miRNA expression using microarrays. The microarray data reveals that there are differences in the expression levels of different miRs, and our studies suggest functional crosstalk between ROS and miR-451 in the regulation of macrophage oxidant stress. Mouse bone marrow-derived macrophages (BMDMs) were obtained from WT (wild type) and p47phox-/- mice. MicroRNAs were isolated by using the mirVana miRNA kit, and a TaqMan rodent microRNA array (consisting of Megaplex RT Primers, Rodent Pool-A, Applied Biosystems) was used for microarray. The array enables quantitation of the expression levels of up to 380 microRNAs and controls. Rodent Pool A contains reverse transcription (RT) primers for 335 and 238 unique microRNAs for mouse and rat, respectively, plus 4 species-specific controls. The data were analyzed on RQ manager software (Qiagen, SA Biosciences) and normalized to the endogenous controls, and analyzed for fold change of miRs in WT compared to p47phox-/-.
Project description:By employing miRCURY LNA™ microRNA Array, we have identified a subset of 21 top miRNAs that are differentially expressed between GM-BMM and M-BMM cells To know the differential expression of miRNA in mouse GM-CSF-induced bone marrow-derived macrophages (GM-BMM) vs. M-CSF-induced BMM (M-BMM)
Project description:MicroRNAs (miRNAs) play a pivotal role in the regulation of hematopoiesis and development of leukemia. Great interest emerged in modulating miRNA expression for therapeutic purposes. In order to identify miRNAs, which specifically suppress leukemic growth of AML with t(8;21), inv(16) or MLL-rearrangement by inducing differentiation, we conducted a miRNA expression profiling in a cohort of 90 cytogenetically characterized, de novo pediatric AML cases. Four miRNAs, specifically downregulated in MLL-rearranged, t(8;21) or inv(16) AMLs, were characterized by their tumor suppressive properties in cell lines representing those respective cytogenetic groups. Among those, forced expression of miR-9 reduced leukemic growth and induced monocytic differentiation of t(8;21) AML cell lines in vitro and in vivo. The tumor suppressive functions of miR-9 were specifically restricted to AML cell lines and primary leukemic blasts with t(8;21). On the other hand, these functions were not evident in AML blasts from patients with MLL-rearrangements. We showed that miR-9 exerts its effects through the cooperation with let-7 to repress the oncogenic LIN28B/HMGA2 axis. Thus, miR-9 is a tumor suppressor-miR which acts in a stringent cell context-dependent manner. In order to identify miRNAs, which specifically suppress leukemic growth of AML with t(8;21) (n=21), inv(16) (n=17) or MLL-rearrangement (n=35) by inducing differentiation, we conducted a miRNA expression profiling in a cohort of 90 cytogenetically characterized, de novo pediatric AML cases, which also included 12 t(15;17) and 5 t(7;12) samples.
Project description:This SuperSeries is composed of the following subset Series: GSE31066: Lipopolysaccharide (LPS) response in macrophages from TLR4-deficient mice GSE31067: Lipopolysaccharide (LPS) response in macrophages from MD-2-deficient mice Refer to individual Series
Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. The gene expression profile of macrophages from C57BL/6 and MD-2-deficient mice following either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 stimulation for 2 hours were compared to PBS-stimulated control cells . In vitro differentiated macrophages from two individual WT and MD-2-deficient mice were cultured and stimulated with agonists separately, comparing the gene expression to PBS-stimulated control cells from the same mouse. Comparisons of PBS-stimulated WT cells to PBS-stimulated MD-2-deficient cells were performed to directly compare basal gene expression in the two genotypes.
Project description:We addressed the potential for global regulation of miRNA biogenesis by BDNF using miRNA arrays that selectively measure mature miRNA, as opposed to pre-miRNA. Hippocampal neurons were treated with BDNF for 30 min in the presence of Actinomycin-D to assess changes due to processing of existing pre-miRNAs rather than new pre-miRNA production. We used Applied Biosystems 7900HT Fast Real-Time PCR system using Taqman Rodent MicroRNA Array A. Data is from three paired BDNF and Mock experiments (1,2,3). Each array (TaqMan) contained 375 rodent miRNA targets of which 195 were detectable in hippocampus in three independent paired experiments.
Project description:We sequenced microRNAs from bone marrow derived macrophages derived from the control (WT) and RBP-J conditional knockout mice (RBP-J KO; Rbpjf/f; LysM Cre). Examination of differential microRNA expression levels induced by TNF as well as regulated by RBP-J in bone marrow derived macrophages.
Project description:The study sought to determine the global miRNA profile of ventricles during early and end-stage hypertrophic cardiomyopathy in a severe double mutant mouse model of the disease. MicroRNA expression profiles of ventricles of transgenic mice with a mutation in both the myosin heavy chain gene (MYH7 Arg403Gln) and cardiac troponin I gene (TNNI3 Ser203Gln) and of non-transgenic mice were determined using Rodent TaqMan Low Density miRNA Arrays A v2.0 (TLDA, Life Technologies). MicroRNA profiles were measured at 10 days of age and 16 days of age, in 3 biological replicates. qRT-PCR analysis of microRNAs of ventricles of three transgenic mice and three non-transgenic mice age 10 days, and three transgenic mice and three non-transgenic mice age 16 days. 450 ng RNA was reverse transcribed, without pre-amplification, using TaqMan MicroRNA Reverse Transcription Kit and Megaplex RT Primers rodent pool A (Life Technologies). Complementary DNA (cDNA) was amplified using a TaqMan rodent microRNA A Array v2.0 (Life Technologies) with TaqMan Universal PCR Master Mix on an ABI 7900HT Sequence Detection System.
Project description:The purpose of this experiment was to determine changes in gene expression by bone marrow-derived macrophages (BMDMs) treated with synthetic human vs. rodent islet amyloid polypeptide (IAPP). Synthetic human IAPP at 15 uM aggregates to form fibrils in vitro, whereas rodent IAPP is non-amyloidogenic. We hypothesized that interaction of macrophages with human IAPP aggregates can activate pro-inflammatory signalling pathways in macrophages, as described for other amyloidogenic peptides. This array includes eight samples from one experiment, with two groups of four replicates each. Each replicate represents BMDMs from one well of a 24-well plate. The control group was treated with 15 uM rat IAPP for 12 hours prior to total RNA extraction; the experimental group was treated with 15 uM human IAPP.
Project description:Macrophages were derived from the bone-marrow of 3 x fl/+ Dicer LysCre +/- (wild-type) and 3 x fl/fl Dicer LysCre +/- mice and stimulated with IL-4 (50ng/mL) for 72h. Total RNA was isolated and analyzed by gene array. In this experiment, we derived Dicer deficient bone-marrow macrophages using Dicer fl/+ LysM-Cre by Dicer fl/+ crossed mice to obtain Dicer fl/fl LysM-cre progeny (and Dicer deficient macrophages). Next, we studied the effects of IL-4 stimulation in macrophage with a deficiency in Dicer/microRNAs.
Project description:Classically activated (M1) macrophages protect from infection but can cause inflammatory disease and tissue damage while alternatively activated (M2) macrophages reduce inflammation and promote tissue repair. Modulation of macrophage phenotype may be therapeutically beneficial and requires further understanding of the molecular programs that control macrophage differentiation. A potential mechanism by which macrophages differentiate may be through microRNA (miRNA), which bind to messenger RNA and post-transcriptionally modify gene expression, cell phenotype and function. The inflammation-associated miRNA, miR-155, was rapidly up-regulated over 100-fold in M1, but not M2, macrophages. Inflammatory M1 genes and proteins iNOS, IL-1b and TNF-a were reduced up to 72% in miR-155 knockout mouse macrophages, but miR-155 deficiency did not affect expression of genes associated with M2 macrophages (e.g., Arginase-1). Additionally, a miR-155 oligonucleotide inhibitor efficiently suppressed iNOS and TNF-a gene expression in wild-type M1 macrophages. Comparative transcriptional profiling of unactivated (M0) and M1 macrophages derived from wild-type and miR-155 knockout (KO) mice revealed an M1 signature of approximately 1300 genes, half of which were dependent on miR-155. Real-Time PCR of independent datasets validated miR-155's contribution to induction of iNOS, IL-1b, TNF-a, IL-6 and IL-12, as well as suppression of miR-155 targets Inpp5d, Tspan14, Ptprj and Mafb. Overall, these data indicate that miR-155 plays an essential role in driving the differentiation and effector potential of inflammatory M1 macrophages. Total RNA was prepared from bone marrow-derived macrophages of miR-155 knockout mice (n=2 independent mice) treated in M0, M1 or M2 conditions (n=2 replicates per condition originating from different mice)