Project description:Sexual dimorphisms are well recognized in various cardiac diseases, including myocardial infarction (MI). MI develops later in women, but once established, it contributes more persistent symptoms and higher mortality than in men. Although mRNA-level sexual dimorphism of MI have been reported, whether miRNA transcriptome also confers such dimorphism remains unknown. Comprehensive understanding of the mRNA- and miRNA-level genetic programs underlying the heart sexual dimorphisms will expectedly improve clinical outcome by facilitating the development of gender specific treatment strategies. Here, by conducting miRNA microarray analysis of human MI samples, we set out to characterize the heart sexual dimorphisms at the level of miRNA transcriptome Human tissue samples, acquired during post-mortem examination and frozen in liquid nitrogen, were provided by the department of pathology, Tokyo Metropolitan Geriatric Hospital after the approval from the ethical committee. Age- and sex-matched cohorts were selected to compare healthy hearts to those with post-MI LV remodeling. Border zone for myocardial infarction was sampled. Total RNA was extracted using Sepasol solution (Sepasol-RNA I super G, nakalai tesque, Japan), and microarray analysis was performed using Affymetrix GeneChip® miRNA 3.0 Arrays
Project description:Sexual dimorphisms are well recognized in various cardiac diseases, including myocardial infarction (MI). MI develops later in women, but once established, it contributes more persistent symptoms and higher mortality than in men. Similar observations have been reported in murine model of MI. Although mRNA-level sexual dimorphism of MI have been reported, whether miRNA transcriptome also confers such dimorphism remains unknown. Comprehensive understanding of the mRNA- and miRNA-level genetic programs underlying the heart sexual dimorphisms will expectedly improve clinical outcome by facilitating the development of gender specific treatment strategies. Here, by conducting miRNA microarray analysis of murine MI model samples, we set out to characterize the heart sexual dimorphisms at the level of miRNA transcriptome The left anterior descending (LAD) coronary artery of mice aged 10 weeks was surgically ligated to create extensive MI. The ventricular septum of the areas at risk of ischemia was sampled on post-operative day 28. Total RNA was extracted using Sepasol solution (Sepasol-RNA I super G, nakalai tesque, Japan), and microarray analysis was performed using Affymetrix GeneChip® miRNA 3.0 Arrays
Project description:Dietary restriction (DR) is the most effective and reproducible intervention to extend lifespan in divergent species1. In mammals, two regimens of DR, intermittent fasting (IF) and caloric restriction (CR), have proven to extend lifespan and reduce the incidence of age-related disorders2. An important characteristic of IF is that it can increase lifespan, even when there is little or no overall decrease in calorie intake2. The molecular mechanisms underlying IF-induced longevity, however, remain largely unknown. Here we establish an IF regimen that effectively extends the lifespan of Caenorhabditis elegans, and show that a nutrient-related signalling molecule, the low molecular weight GTPase Cel-Rheb, has a dual role in lifespan regulation; Cel-Rheb is required for the IF-induced longevity, whereas inhibition of Cel-Rheb mimics the CR effects. We also show that Cel-Rheb exerts its effects in part via the insulin/IGF-like signalling effector DAF-16 in IF, and that Cel-Rheb is required for fasting-induced nuclear translocation of DAF-16. We find that HSP-12.6, a DAF-16 target, functions to mediate the IF-induced longevity. Furthermore, our analyses demonstrate that most of fasting-induced upregulated genes require Cel-Rheb function for their induction, and that Cel-Rheb/Cel-TOR signalling is required for the fasting-induced downregulation of an insulin-like peptide, INS-7. These findings identify the essential role of signalling via Cel-Rheb in IF-induced longevity and gene expression changes, and suggest a molecular link between the IF-induced longevity and the insulin/IGF-like signalling pathway. Experiment Overall Design: We examined fasting-induced changes of the gene expression profiles in Caenorhabditis elegans. We performed the genome-wide analysis by using Affymetrix GeneChip oligonucleotide microarrays, and examined the effect of downregulation of Cel-Rheb and Cel-TOR by RNAi on the expression profiles. Five independent experiments were performed with wild type N2. Synchronized worms under six conditions (control-fed, control-fasting, Rheb RNAi-fed, Rheb RNAi-fasting, TOR RNAi-fed, and TOR RNAi-fasting) were collected and frozen with liquid nitrogen at day 4 of adulthood. Total RNA was extracted with Sepasol(R)-RNA â Super (Nacalai tesque), and purified with RNeasy Mini Kit (Qiagen), according to manufactureâs instructions. Synthesis of cDNA, in vitro transcription and biotin labelling cRNA, and hybridization to the C. elegans Genome Array (Affymetrix) were performed according to Affymetrix protocols. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner. Scanned chip images were analyzed with GeneSpring GX 7.3.1 (Agilent Technologies).
Project description:PTIP (Pax2 transactivation domain-interacting protein) is a nuclear protein containing six BRCT domains. It has been shown that PTIP affects gene expression by controlling the activity of the transcription factor Pax2 and histone H3 lysine 4 methyltransferase complexes. In addition to its role in transcriptional regulation, PTIP has been implicated in DNA damage response. To ask if the depletion of PTIP affects the expression level of genes encoding DNA damage response factors , we compared the whole transcripts between wild-type and PTIP deficient chicken DT40 B cell lines. The total RNAs were isolated from wild-type and PTIP deficient cells (PTIP-/-/-) using Sepasol®-RNA I (Nacalai tesque, Japan). The gene expression profiles were examined using Genechip® Chicken Genome Array (Affymetrix Cat #900590), by GeneticLab co. Ltd. Japan, following Genechip® protocol.
Project description:We used microarrays to evaluate the effect of SRPIN803 on gene expression in ARPE-19 cells. ARPE-19 cells were treated with SRPIN803 (10 uM) or the negative control (0.1% DMSO) for 4 hours for Total RNA isolation and hybridization on Microarray.
Project description:We recently showed that exosomes from primary AML cells and cell lines have potent regulatory capacity and we hypothesized that leukemia cell exosome trafficking might account for the suppression of residual hematopoietic stem and progenitor cells (HSPC) in the leukemic niche. Here we studied Molm-14 cells in in vitro experiments using purified exosomes under carefully calibrated low-oxygen conditions. This approach revealed the active regulation of stromal- and hematopoietic progenitor cell function. Systematic analysis of AML exosomes identified a panel of differentially enriched hypoxia-responsive miRNAs, including miR-210, -155, and -146a. We cultured cells under normoxic and hypoxic conditions. Exosomes were released from the parental cells and captured. Total RNA was isolated from both exosomes and cells. Please note that the sample numbers (in the titles) do differ from pair identification (i.e. pair ID) as the pair is only in reference to the paired-t test and parental vs. released pairs.
Project description:Ischemia reperfusion (I/R) injury is an unavoidable event occurring during heart transplantation, leading to graft failures and lower long-term survival rate of the recipient. microRNAs are major regulators of genes. IR causes apoptosis/death of cardiomyocytes, resulting from up-regulation of apoptotic genes and down-regulation of anti-apoptotic genes which are regulated by microRNA. We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process. We perserved donor hearts with university of Wisconsin solution for 18h at 4C degree before being implanted into recipients to create ischemia reperfusion injury. The preserved hearts were implanted into a syngeneic recipient mice. 24h after transplantation, heart grafts were harvested for microRNA extraction.
Project description:Our study was designed to identify plasma miRNAs specific for rheumatoid arthritis (RA) by a comprehensive array approach. We performed a array-based miRNA analysis on plasma samples from three RA patients and three healthy controls (HCs). TaqMan Low-Density Array (TLDA) using human miRNA version 3.0A and version 2.0B cards (Applied Biosystems) were applied to examine the global change of miRNA expression levels in plasma from patients with RA and healthy controls. A total of 756 mature miRNA updated in the Sanger miRBase v.15.0 were quantified according to the manufacturer's instructions as previously described. Normalization was carried out with the average Ct value of all miRNAs. Relative quantification of miRNA expression was calculated with the 2−ΔΔCt Ct method. The data was presented as log10 of the relative quantity of each miRNA.
Project description:Zinc-finger genes Fezf1 and Fezf2 encode transcriptional repressors. Fezf1 and Fezf2 are expressed in the early neural stem/progenitor cells and control neuronal differentiation in mouse dorsal telencephalon. We compared gene expression profiles of rostral forebrains, which contain the telencephalon and the rostral part of the diencephalon, from embryonic day (E) 9.5, E10.5, and E12.5 wild-type control and Fezf1-/- Fezf2 -/- mouse embryos. The forebrain rostral to the caudal limit of the lateral ventricles was isolated manually from E9.5, E10.5, and E12.5 wild-type and Fezf1-/- Fezf2-/- mice. Total RNAs were isolated by Separsol-RNA I and were used for microarray analyses.