MiR-206 modulates the pathogenesis of muscular dystrophy of mdx mice
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ABSTRACT: In response to skeletal muscle injury, adult myogenic stem cells, known as satellite cells, are activated and undergo proliferation and differentiation to regenerate new muscle fibers. The skeletal muscle-specific microRNA, miR-206, is up-regulated in satellite cells following muscle injury, but its role in muscle regeneration has not been defined. Here we show that skeletal muscle regeneration in response to cardiotoxin injury is impaired in mice lacking miR-206. Loss of miR-206 also accelerates and exacerbates the dystrophic phenotype of mdx mice, a model for Duchenne muscular dystrophy. MiR-206 promotes satellite cell differentiation and fusion to form multinucleated myofibers by suppressing a collection of negative regulators of myogenesis. Our findings reveal an essential role for miR-206 in satellite cell differentiation during skeletal muscle regeneration and as a modulator of Duchenne muscular dystrophy. total RNA obtained from TA muscle of mdx and 3 miR-206 KO; mdx mice at 3 months of age.
Project description:In response to skeletal muscle injury, adult myogenic stem cells, known as satellite cells, are activated and undergo proliferation and differentiation to regenerate new muscle fibers. The skeletal muscle-specific microRNA, miR-206, is up-regulated in satellite cells following muscle injury, but its role in muscle regeneration has not been defined. Here we show that skeletal muscle regeneration in response to cardiotoxin injury is impaired in mice lacking miR-206. Loss of miR-206 also accelerates and exacerbates the dystrophic phenotype of mdx mice, a model for Duchenne muscular dystrophy. MiR-206 promotes satellite cell differentiation and fusion to form multinucleated myofibers by suppressing a collection of negative regulators of myogenesis. Our findings reveal an essential role for miR-206 in satellite cell differentiation during skeletal muscle regeneration and as a modulator of Duchenne muscular dystrophy.
Project description:To identify microRNAs impacting estrogen receptor ERα expression in breast cancer, we have screened ER-positive breast cancer cells with a library of pre-miRs, and systematically monitored the ERα expression by protein lysate microarrays. There was a significant enrichment of the in silico predicted ERα targeting microRNAs among the hits. The most potent pre-miRs miR-18a/b, miR-193b, miR-206, and miR-302c, were confirmed to directly target ERα and to repress estrogen-responsive genes. The effect of miRNA overexpression on gene expression profile of MCF-7 cells was studied. Furthermore, miR-18a and miR-18b showed increased expression in ERα-negative as compared to ERα-positive clinical tumors. In summary, we present systematic and direct functional and correlative clinical evidence on microRNAs inhibiting ERα signaling in breast cancer. MCF-7 cells (300 000 per well on 6-well plates) were transfected with an siRNA for ERα or with Ambion pre-miR� constructs for miR-18a, miR-193b, miR-206, miR-302c, or pre-miR negative control #1 (scrambled pre-miR) at 20 nM, and incubated for 24h.
Project description:Early reperfusion of ischemic cardiac tissue remains the most effective intervention for improving clinical outcome following myocardial infarction. However, abrupt increases in intracellular Ca2+ during myocardial reperfusion cause cardiomyocyte death and consequent loss of cardiac function, referred to as ischemia/reperfusion (IR) injury. Cardiac IR is accompanied by dynamic changes in expression of microRNAs (miRNAs), which inhibit specific mRNA targets. miR-214 is up-regulated during ischemic injury and heart failure in mice and humans, but its potential role in these processes is unknown. We show that genetic deletion of miR-214 in mice causes loss of cardiac contractility, increased apoptosis, and excessive fibrosis in response to IR injury. The microarray contains 6 samples, each containing cDNA pooled from 3 mice per group. There are no replicates. The array was designed to make 3 different pairwise comparisons between the following: P14 WT and miR-214 KO hearts; adult WT and miR-214 KO skeletal muscle; adult WT and miR-214 KO hearts
Project description:Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, and their role in the disease remains unresolved. Here, we demonstrate that loss of a muscle-specific protein, Klhl40, results in a nemaline-like myopathy in mice that closely phenocopies the muscle abnormalities observed KLHL40 deficient patients. We show that Klhl40 dynamically localizes to the sarcomere I-band and A-band and binds to Nebulin (Neb), a protein frequently implicated in NM, as well as a putative thin filament protein, Lmod3. Klhl40 belongs to the BTB-BACK-Kelch (BBK) family of proteins, some of which have been previously shown to promote degradation of their substrates. In contrast, we find that Klhl40 promotes stability of Neb and Lmod3 and blocks Lmod3 ubiquitination. Accordingly, loss of Klhl40 reduces Neb and Lmod3 protein in skeletal muscle of mice and KLHL40 deficient patients. Because loss of sarcomere thin filament proteins is a frequent cause of NM, our data establishes a possible molecular basis for NM in KLHL40 deficient patients by establishing a novel pro-stability function of Klhl40 for Neb and Lmod3. Total RNA was harvested from quadriceps muscle of three Klhl40 WT (control) and three Klhl40 KO mice. Each KO mouse was sacrificed with a corresponding WT littermate. Tissues were also taken at 0 days of age to minimize confounding gene changes occurring due to malnourishment as the phenotype worsens.
Project description:miR-127 is an imprinted microRNA on mouse chromosome 12, strongly expressed during late embryogenesis and known regulator of placental gene Rtl1. miR-127-knockout (KO) mice appear phenotypically normal. An Illumina beadchip whole genome microarray experiment was carried out on embryonic stage 18.5 (E18.5) mice with a deletion in the miR-127 gene, and compared with wild type (WT) mice. Three tissues with varying expression of miR-127 were analysed: brain, skin and muscle. For each tissue (brain, skin, muscle) and genotype (WT or miR-127 KO), total RNA from 15 different embryos was extracted. These RNA samples were divided into three pools of five, to make three biological replicates. Each biological replicate was applied to two separate Illumina Mouse WG-6 v2.0 beadchips, to make two technical replicates.
Project description:The experiment was deigned to identify the genes which get altered after the over expression of hsa-miR-128 in HEK293T cells. The HEK293T cells plated in 6-well plate were transfected with 4 micrograms of cloned miR-128 (p128) in three biological replicates. Biological triplicates of 2 samples were used viz. control HEK293T cells (c), HEK293T cells transfected with 4 micrograms of the cloned hsa-miR-128(t).
Project description:Mouse models have proven invaluable for understanding erythropoiesis. Here, we describe an autosomal recessive inherited anemia in the mouse mutant hem6. Hematologic and transplantation analyses revealed a mild, congenital, hypochromic, microcytic anemia intrinsic to the hematopoietic system that is associated with a decreased red blood cell zinc protoporphyrin to heme ratio, indicative of porphyrin insufficiency. Iron uptake experiments showed that hem6 reticulocytes are defective in heme production, but not cellular iron uptake defects. Male hem6 mice are infertile due to defects in sperm structure and motility. Through positional cloning and BAC complementation, we identified the gene responsible for the hem6 anemia. We hypothesized that the relative deficiency in erythroid-specific mRNAs in hem6 reticulocytes might be due to decreased mRNA stability. Indeed, serial microarray analysis of reticulocytes aged in vitro showed that numerous, abundantly expressed erythroid-specific transcripts decayed at faster rates in hem6 reticulocytes compared to control reticulocytes. Furthermore, these mRNAs also have progressively shorter poly (A) tails, suggesting a mechanism for the increased rate of decay. Keywords: serial time points Reticulocyte rich blood were collected and cultured ex vivo for 24 hours, samples were collected at 0, 12,24 hours for microarray analysis. There are 3 wild type (wt) biological replicates and 5 mutant (mut) biological replicates in each time point.
Project description:Human pluripotent stem cells (hPSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of hPSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However the effective use of hPSCs for cell therapy has lagged far behind. While mouse PSC-derived DA neurons have shown efficacy in models of Parkinson’s disease, DA neurons derived from human PSCs generally display poor in vivo performance. There are also considerable safety concerns for hPSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor plate-based strategy for the derivation of human DA neurons that efficiently engraft, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor plate precursors are derived from hPSCs in days following exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signaling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive in vitro molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of hPSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in PD animal models in three host species. Long-term engraftment in 6-OHDA-lesioned mouse and rats demonstrates robust survival of midbrain DA neurons, complete restoration of amphetamine-induced rotation behavior and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into Parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models tested indicate considerable promise for the development of cell based therapies in PD. Differentiated hESC with three conditions (LSB, LSB/S/F8, LSB/S/F8/CHIR) were subjected to RNA extraction in specific timepoint (day 0, 1, 3, 5, 7, 11, 13, 25) and hybridization on Illumina microarrays. Each sample has 3 or 4 biological repeats. Based on previous study* of dual SMAD inhibition neural induction, we developed new midbrain dopamine neuron protocol. It depends on time specific treatment of below factors (LSB/S/F8/CHIR): L (LDN193189 (BMP inhibitor) , day 0-11), SB (SB431542 (TGF-b signal inhibitor), day 0-5), S (SHH + Purmorphamine (Smo agonist), day 1-7), F8 (FGF8, day 1-7) and CHIR (CHIR99021 (GSK3b inhibitor), day 3-13) LSB and LSB/S/F8 are limited control conditions of dual SMAD only (LSB) or traditional patterning with Sonic and FGF (LSB/S/F8) *Chambers,S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275-280 (2009).
Project description:Biotin labelled miR-206 mimics ('06") and miR-Control mimics ("NC") were used to indentify putative miRNA transcript targets in FAPs form skeletal muscle