DOCSLIB.ORG

Structural Elucidation of Mrna(Sirt1)- Microrna 34A Complex

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Structural Elucidation of Mrna(Sirt1)- Microrna 34A Complex Structural elucidation of mRNA(Sirt1)- microRNA 34a complex Mona Farshchian Master thesis in Technology and learning, degree project for the study program Master of Science in Engineering and of Education, Stockholm 2015. Degree Project in Technology and Learning of 30 ECTS in the program Master of Science in Engineering and of Education, Degree Program in Mathematics and Chemical Science, Royal Institute of Technology, KTH, and Stockholm University, SU Mona Farshchian: Structural elucidation of mRNA(Sirt1)-microRNA 34a complex, Stockholm 2015 MAIN SUPERVISOR Peter Savolainen, Associate Professor, BIO, Kungliga Tekniska Högskolan SECONDARY SUPERVISOR Åsa Julin-Tegelman, Assistant Professor, Education, Stockholm University EXTERNAL SUPERVISOR Katja Petzold, Assistant Professor, Medical Biochemistry and Biophysics, Karolinska Institutet EXAMINER Joakim Lundeberg, Professor, BIO, Kungliga Tekniska Högskolan 2 Abstract The aim of this thesis is to understand RNA-RNA interactions steering cellular functions, as in the case of this thesis the structure of mRNA(Sirt1) in complex with microRNA-34a (miR-34a). MiR-34a regulates the cancer protein p53 via Sirt1 modulation. This work will be the basis for future drug design and the understanding of misguided regulation in cancer. The miR-34a binds to the mRNA(Sirt1) 3’ untranslated region (3’-UTR) and will either inhibit the translation of the protein Sirtuin 1 by capturing its mRNA or by degrading it. p53, a key activator of miR-34a, prevents cancer development by inducing programmed cell death (apoptosis) on cells with DNA damage. In contrast, the protein Sirtuin 1 (Sirt1) has been shown to help cells with DNA damage to survive by down regulating the activity of protein p53 and will therefore increase the risk of cancer development. Studying the interaction between the mRNA(Sirt1) and miR-34a can present valuable information on the structure of the complex as well as the mode miR-34a uses to inhibit translation of mRNA(Sirt1) leading to down regulation of protein Sirtuin 1 and therefore prevent cancer development. For the elucidation of this question different biochemical and biophysical methods were applied, such as in vitro transcription, gel electrophoresis, RNA purification with gel, crush & soak and Cicular Dichroism (CD) melting studies. For this thesis work, the target sequence in mRNA(Sirt1) was optimized and purified so melting studies could be carried out. For future structural characterization using Nuclear Magnetic Resonance (NMR) studies with the miR-34a also produced in the lab. The mRNA(Sirt1) target sequence was produced and purified with the final yield of 0.02%. The results show that the sequence is highly ATP dependent and suggest the ratio between the nucleotides ATP/CTP to be 1:2. Low yield of purified mRNA(Sirt1) was received and still contained some impurities, which imply that another method than crush & soak should be used when purifying. The results, indicate that High-Preformance Liquid Chromatography (HPLC) might be a better solution for the pufication process. The melting profiels done on mRNA(Sirt1) show that the secondary structures decrease with an increase in temperature. Accroding to the results, the mRNA(Sirt1) sequence is folded in room temperature, though not very stable. The wavelength which provided the best resolution was at 268 nm and the melting point of mRNA(Sirt1) was determined to 44 °C. This thesis also contains an educational part, where an educational material was provided and testing was conducted for the subject Chemistry 2 for students age 18 and the material was evaluated with qualitative methods together with pedagogical methods. The study showed that the student can develope the different abilities stated in the curriculum with the material created. The results also showed that the students preferably choose cultural arguments when dicussing socio scientific question, rather than economical, democratic or utility arguments. Keywords: mRNA(Sirt1), miR-34a, in vitro transcription, gel electrophoresis, CD spectroscopy, NMR, cancer regulation via p53, HPLC 3 Sammanfattning Syftet med studien är att förstå RNA-RNAinteraktioner som styr cellulära funktioner, i detta fall mRNA(Sirt1) i komplex med microRNA-34a (miR-34a). MiR-34a reglerar cancerproteinet p53 via modulation av Sirt1. Detta arbete kommer lägga grund för framtida läkemedelsdesign vid reglering av cancer. MiR-34a binder till den 3’ otranslerade regionen i mRNA(Sirt1) och hämmar antingen translationen av protein Sirtuin 1 (Sirt1) genom att fånga dess mRNA eller genom att försämra det. p53 förhindrar utvecklingen av cancer genom att framkalla programmerad cell död (apoptosis) av celler med skadat DNA. Det har visats att proteinet Sirtuin 1 hjälper celler med skadat DNA att överleva, genom att sänka aktiviteten av p53. På så vis ökar risken för utveckling av cancer. Genom att studera interaktionen mellan mRNA(Sirt1) och miR-34a kan värdefull information kring komplexets struktur fås. Samt hur miR-34a hämmar translationen av mRNA(Sirt1), vilket leder till minskad aktivitet av protein Sirt1. För att klarlägga denna fråga har olika biokemiska och biofysiska metoder använts, såsom in vitro transkription, gelelektrofores, RNA rening med gel och Circular Dichroism (CD). För detta arbete har målsekvensen i mRNA(Sirt1) optimerats och renats så CD smältstudier med kunde genomföras. Resultatet visar att mRNA(Sirt1) sekvensen renats med ett utbyte på 0.02 %. Sekvensen är beroende av ATP och förhållandet mellan ATP/CTP nukleotider bör vara 1:2. Resutatet visar på ett lågt utbyte som visar på att High-Performance Liquid Chromatography (HPLC) kan vara en bättre metod än Crush & soak för reningen av mRNA(Sirt1). Ur de smältprofiler som gjorts visade det sig att de sekundära strukturerna av mRNA(Sirt1) minskade med ökande temperatur. I enlighet med resultaten visar det att mRNA(Sirt1) är veckat i rumstemperatur men är inte stabil. Den bästa upplösningen erhölls vid 268 nm och mRNA(Sirt1) har en smältpunkt runt 44 °C. Detta arbete innehåller även ett utbildningskapitel, där ett utbildningsmaterial har skapats och testats på 18-åriga kemi 2 studenter i åldern 18 år. Materialet har utvärderats med hjälp av kvalitativa metoder tillsammans med pedagogiska metoder. Studien visade att de flesta förmågorna för kemi 2 kan utvecklas med hjälp av denna typ samhällsfrågor i det naturvetenskapliga klassrummet (SNI-fall) förutom förmågan att planera och genomföra experiment. Det argument som eleverna helst väljer att använda då de diskuterar det skapade SNI-fallet är Kulturargument och det minst använda är Demikratiargument. Nyckelord: mRNA(Sirt1), miR-34a, in vitro transkription, gel elektroforesis, CD spektroskopi, NMR, cancer regulering via p53, HPLC 4 Acknowledgements I would like to start by thanking the Petzold lab for giving me the opportunity to write and complete my thesis. Special thanks to Katja Petzold and Lorenzo Baronti for their guidance and support throughout this project. I would also like to thank my family and my fiancé Peyman Eshtiagh for always supporting me and encouraging me towards my goals. I would also like to thank my KTH supervisor Peter Savolainen and SU supervisor Åsa Julin-Tegelman for all the support, along with everyone else who contributed to this thesis. 5 Abstract 3 Sammanfattning 4 Acknowledgements 5 1. INTRODUCTION 8 1.1 Introduction and background 8 1.2 Research questions addressed in Masters Thesis 9 1.3 Abbreviations and terms 10 1.6 LITERATURE STUDY 12 1.6.1 The human cell 12 1.6.2 The transfer of genetic information 13 1.6.3 The transcription reaction 14 1.6.4 Cancer 16 1.6.5 MicroRNA-mRNA interactions 16 1.6.6 MicroRNA 17 1.6.7 Sirt1-p53 regulation 18 1.6.8 Circular Dichroism 20 2. METHOD 21 2.1 Introduction 21 2.2 RNA by In Vitro Transcription 21 2.2.1 Annealing reaction 21 2.2.2 in vitro transcription by T7 - optimization 22 2.3 Polyacrylamide Gel Electrophoresis 25 2.3.1 Gel staining with Ethidium Bromide & UV detection 26 2.4 Large Scale Transcription reaction 26 2.5 mRNA(Sirt1) purification 27 2.6 Circular Dichroism UV melting studies 29 3. RESULTS & Discussion 31 3.1 Optimization of mRNA(Sirt1) 31 3.2 Large scale transcription reaction of mRNA(Sirt1) 35 3.2.1 Verifying with RNAse inhibitor and labeled nucleotides 35 3.2.2 Verifying with Pyruvate Phosphates and longer reaction time 36 3.2 Purification 36 3.4 CD melting studies of mRNA(Sirt1)-miR-34a complex 39 4. CONCLUSION 42 5. FURTHER RESEARCH 43 6 6. UTBILDNINGSKAPITEL 44 6.1 Inledning 44 6.2 Litteraturstudie 45 6.2.1 Forskning i klassrummet 45 6.2.2 Ämnesplaner skolverket 46 6.2.5 Samhällsfrågor med naturvetenskapligt innehåll 47 6.3 Syfte och frågeställning 47 6.4 Metod 48 6.4.1 Datainsamling 48 6.4.2 Urval och kvalitetsredovisning 48 6.5 Resultat 49 6.5.1 Förmågor som tränas med hjälp av SNI-fallet utifrån elevperspektiv 49 6.5.2 Förmågor som tränas med hjälp av SNI-fallet utifrån lärarperspektiv 50 6.5.3 Argument som elever använder i diskussion om frågan genmanipulation med hjälp av SNI-fallet 50 6.6 Analys & Diskussion 52 6.7 Slutsats 53 6.8 Vidare forskning 53 7. REFERENCE 54 7.1 Articles and books 54 7.4 Graphics 57 8. APPENDIX A - Lab protocol 58 9. APPENDIX B - Calculations 62 10. APPENDIX C - Educational material 65 Bilaga 1. Lärarhandledning 65 Bilaga 2 Enkätundersökning 70 Bilaga 3 Kvalitativ intervju 71 7 1. INTRODUCTION This part of the thesis explains the purpose and the background of the project, how it has been done and why the project has been carried out. 1.1 Introduction and background This project is a master thesis in the field of Technology and Learning for the program Master of Science in Engineering and of Education at the Royal Institute of Technology and at Stockholm University. The project is in cooperation with the Petzold group at the Molecular Structural Biology unit at the department of Medical Biochemistry and Biophysics at Karolinska Institute.
Load more

Recommended publications
  • Sirna Interference Or Mirna Mimicry Peipei Wang1,2; Yue Zhou, Ph.D.1,2; Arthur M
    Theranostics 2021, Vol. 11, Issue 18 8771 Ivyspring International Publisher Theranostics 2021; 11(18): 8771-8796. doi: 10.7150/thno.62642 Review Effective tools for RNA-derived therapeutics: siRNA interference or miRNA mimicry Peipei Wang1,2; Yue Zhou, Ph.D.1,2; Arthur M. Richards, M.D., Ph.D.1,2,3 1. Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, 117599 Singapore. 2. Department of Medicine, National University Health System, 119228 Singapore. 3. Christchurch Heart Institute, Department of Medicine, University of Otago Christchurch, New Zealand. Corresponding author: Peipei Wang, MD, PhD, Cardiovascular Research Institute, Department of Medicine, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Centre for Translational Medicine, MD6, #08-01, 14 Medical Drive, Singapore 117599. Phone: (65) 81613586; Fax: (65) 6775-9715; E-mail: [email protected]. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2021.05.12; Accepted: 2021.07.30; Published: 2021.08.11 Abstract The approval of the first small interfering RNA (siRNA) drug Patisiran by FDA in 2018 marks a new era of RNA interference (RNAi) therapeutics. MicroRNAs (miRNA), an important post-transcriptional gene regulator, are also the subject of both basic research and clinical trials. Both siRNA and miRNA mimics are ~21 nucleotides RNA duplexes inducing mRNA silencing. Given the well performance of siRNA, researchers ask whether miRNA mimics are unnecessary or developed siRNA technology can pave the way for the emergence of miRNA mimic drugs.
    [Show full text]
  • Activation of Nuclear Factor-Kappa B Signalling Promotes Cellular Senescence
    Oncogene (2011) 30, 2356–2366 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Activation of nuclear factor-kappa B signalling promotes cellular senescence E Rovillain1, L Mansfield1, C Caetano1, M Alvarez-Fernandez2, OL Caballero3, RH Medema2, H Hummerich4 and PS Jat1 1Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK; 2Department of Medical Oncology, University Medical Center, Utrecht, The Netherlands; 3Ludwig Institute for Cancer Research Ltd, New York, NY, USA and 4MRC Prion Unit, UCL Institute of Neurology, London, UK Cellular senescence is a programme of irreversible cell of intrinsic and extrinsic stimuli including progressive cycle arrest that normal cells undergo in response to shortening of telomeres or changes in telomeric struc- progressive shortening of telomeres, changes in telomeric ture or other forms of genotoxic stress such as oncogene structure, oncogene activation or oxidative stress. The activation, DNA damage or oxidative stress resulting in underlying signalling pathways, of major clinicopatholo- a DNA damage response and growth arrest via gical relevance, are unknown. We combined genome-wide activation of the p53 tumour suppressor (Ben-Porath expression profiling with genetic complementation to and Weinberg, 2005; Campisi and d’Adda di Fagagna, identify genes that are differentially expressed when 2007; Adams, 2009). Activation of p53 induces p21CIP1, conditionally immortalised human fibroblasts undergo which prevents cyclin/cdk complexes from phosphor- senescence upon activation of the p16-pRB and p53-p21 ylating the RB family of proteins, thereby activating the tumour suppressor pathways. This identified 816 up and pRB tumour suppressor pathway (Ben-Porath and 961 downregulated genes whose expression was reversed Weinberg, 2005; Campisi and d’Adda di Fagagna, when senescence was bypassed.
    [Show full text]
  • Open Full Page
    Published OnlineFirst July 22, 2014; DOI: 10.1158/1535-7163.MCT-14-0209 Molecular Cancer Large Molecule Therapeutics Therapeutics Systemic Delivery of a miR34a Mimic as a Potential Therapeutic for Liver Cancer Christopher L. Daige, Jason F. Wiggins, Leslie Priddy, Terri Nelligan-Davis, Jane Zhao, and David Brown Abstract miR34a is a tumor-suppressor miRNA that functions within the p53 pathway to regulate cell-cycle progression and apoptosis. With apparent roles in metastasis and cancer stem cell development, miR34a provides an interesting opportunity for therapeutic development. A mimic of miR34a was complexed with an amphoteric liposomal formulation and tested in two different orthotopic models of liver cancer. Systemic dosing of the formulated miR34a mimic increased the levels of miR34a in tumors by approximately 1,000-fold and caused statistically significant decreases in the mRNA levels of several miR34a targets. The administration of the formulated miR34a mimic caused significant tumor growth inhibition in both models of liver cancer, and tumor regression was observed in more than one third of the animals. The antitumor activity was observed in the absence of any immunostimulatory effects or dose-limiting toxicities. Accumulation of the formulated miR34a mimic was also noted in the spleen, lung, and kidney, suggesting the potential for therapeutic use in other cancers. Mol Cancer Ther; 13(10); 2352–60. Ó2014 AACR. Introduction with orthotopic Hep3B and HuH7 liver cancer xenografts. The development of lipid nanoparticle technologies Systemic delivery of the encapsulated miR34a mimic that provide efficient delivery of RNA oligonucleotides reduced the expression levels of several miR34a target to hepatocytes and liver tumors (1) has created an oppor- mRNAs and caused significant growth inhibition and tunity for RNAi-based therapies.
    [Show full text]
  • Mirnas As Biomarkers for Prostate Cancer Progression
    Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2015 MIRNAS AS BIOMARKERS FOR PROSTATE CANCER PROGRESSION Gene C. Clark Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Diagnosis Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/3954 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. MIRNAS AS BIOMARKERS FOR PROSTATE CANCER PROGRESSION A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Virginia Commonwealth University By Gene Chatman Clark B. S. 2012, Virginia Polytechnic Institute Director: Zendra E. Zehner Professor, Department of Biochemistry Virginia Commonwealth University Richmond, Virginia July 22, 2015 Acknowledgement Firstly, I would like to thank the many people here at VCU have inexplicably taken a chance on me. This includes, but could not possibly be limited to, Dr. Kimberly Jefferson who let me work in her lab when I was just a non-degree seeking student, Dr. Shirley Taylor who successfully encouraged me to pursue my love of research, and Dr. Louis DeFelice who suggested that I apply to the Pre-Medical Certificate Program here at VCU. I would also like to thank my amazing lab mates, Qianni Wu and Rhonda Daniel, whose insight and support were invaluable to this project. I have been lucky to have had them as partners and as friends.
    [Show full text]
  • Evaluation of Pre-Mir-34A Rs72631823 Single Nucleotide Polymorphism in Triple Negative Breast Cancer: a Case-Control Study
    www.oncotarget.com Oncotarget, 2018, Vol. 9, (No. 97), pp: 36906-36913 Research Paper Evaluation of pre-mir-34a rs72631823 single nucleotide polymorphism in triple negative breast cancer: A case-control study Despoina Kalapanida1, Flora Zagouri1, Maria Gazouli2,3, Eleni Zografos2,3, Constantine Dimitrakakis4, Spyridon Marinopoulos4, Aris Giannos4, Theodoros N. Sergentanis1, Efstathios Kastritis1, Evangelos Terpos1 and Meletios-Athanasios Dimopoulos1 1Department of Clinical Therapeutics, Alexandra Hospital, Medical School, University of Athens, Athens, Greece 2Department of Basic Medical Sciences, Laboratory of Biology, University of Athens School of Medicine, Athens, Greece 3Laboratory of Cell and Gene Therapy, Biomedical Research Foundation of the Academy of Athens, Athens, Greece 4Department of Obstetrics and Gynaecology, Alexandra Hospital, Medical school, University of Athens, Athens, Greece Correspondence to: Flora Zagouri, email: [email protected] Keywords: SNPs; pre-miR-34a; miRNA; triple negative breast cancer; biomarker Received: September 13, 2018 Accepted: November 03, 2018 Published: December 11, 2018 Copyright: Kalapanida et al. This is an open-access article distributed under the terms of the Creative Commons Attribution Li- cense 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Aim: The purpose of this study is to evaluate the role of pre-miR34a rs72631823 as potential risk factor and/or prognostic marker in patients with triple negative breast cancer. Methods: 114 samples of DNA from paraffin embedded breast normal tissues of patients with triple negative breast cancer and 124 samples of healthy controls were collected and analyzed for pre-miR34a rs72631823 polymorphism. Results: Pre-miR34a rs72631823 A allele was associated with increased TNBC risk both in univariate and multivariate analysis.
    [Show full text]
  • In Silico Analysis of Polymorphisms in Micrornas That Target Genes Affecting Aerobic Glycolysis
    Original Article Page 1 of 8 In silico analysis of polymorphisms in microRNAs that target genes affecting aerobic glycolysis Padmanaban S. Suresh1, Thejaswini Venkatesh2, Rie Tsutsumi3 1Department of Biosciences, Mangalore University, Mangalagangotri, Mangalore 574 199, Karnataka, India; 2Nitte University Centre for Science Education and Research, Nitte University, Derlakatte, Mangalore, Karnataka, India; 3Department of Nutrition and Metabolism, Institute of Biomedical Science, Tokushima University, Tokushima, Japan Contributions: (I) Conception and design: PS Suresh, T Venkatesh; (II) Administrative support: None; (III) Provision of study materials or patients: None; (IV) Collection and assembly of data: PS Suresh; (V) Data analysis and interpretation: PS Suresh; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. Correspondence to: Padmanaban S. Suresh. Department of Biosciences, Mangalore University, Mangalagangotri, Mangalore 574 199, Karnataka, India. Email: [email protected]. Background: Cancer cells preferentially metabolize glucose through aerobic glycolysis, an observation known as the Warburg effect. Recently, studies have deciphered the role of oncogenes and tumor suppressor genes in regulating the Warburg effect. Furthermore, mutations in glycolytic enzymes identified in various cancers highlight the importance of the Warburg effect at the molecular and cellular level. MicroRNAs (miRNAs) are non-coding RNAs that posttranscriptionally regulate gene expression and are dysregulated in the pathogenesis of various types of human cancers. Single nucleotide polymorphisms (SNPs) in miRNA genes may affect miRNA biogenesis, processing, function, and stability and provide additional complexity in the pathogenesis of cancer. Moreover, mutations in miRNA target sequences in target mRNAs can affect expression. Methods: In silico analysis and cataloguing polymorphisms in miRNA genes that target genes directly or indirectly controlling aerobic glycolysis was carried out using different publically available databases.
    [Show full text]
  • Exosomal Micrornas: Pleiotropic Impacts on Breast Cancer Metastasis and Their Clinical Perspectives
    biology Review Exosomal microRNAs: Pleiotropic Impacts on Breast Cancer Metastasis and Their Clinical Perspectives Li-Bo Tang 1,2,† , Shu-Xin Ma 3,†, Zhuo-Hui Chen 2, Qi-Yuan Huang 1,2 , Long-Yuan Wu 1,4, Yi Wang 1, Rui-Chen Zhao 1,3 and Li-Xia Xiong 1,5,* 1 Department of Pathophysiology, Basic Medical College, Nanchang University, Nanchang 330006, China; [email protected] (L.-B.T.); [email protected] (Q.-Y.H.); [email protected] (L.-Y.W.); [email protected] (Y.W.); [email protected] (R.-C.Z.) 2 Second Clinical Medical College, Nanchang University, Nanchang 330006, China; [email protected] 3 Queen Mary School, Jiangxi Medical College, Nanchang University, Nanchang 330006, China; [email protected] 4 First Clinical Medical College, Nanchang University, Nanchang 330006, China 5 Jiangxi Province Key Laboratory of Tumor Pathogenesis and Molecular Pathology, Nanchang 330006, China * Correspondence: [email protected]; Tel.: +86-791-8636-0556 † These authors contributed equally to this work. Simple Summary: This review has comprehensively summarized the most recent studies in the last few years about exosomal microRNAs on metastasis of breast cancer (BC), systematically outlined and elucidated the pleiotropic roles that exosomal microRNAs take, and discussed the specific underlying mechanisms related. Besides, we also clearly demonstrate the clinical implications of exosomal microRNAs in various aspects, including early-stage discovery of BC, systematic and Citation: Tang, L.-B.; Ma, S.-X.; Chen, targeted therapy, and the selection of anti-cancer chemo-agents. This review clarifies the scope and Z.-H.; Huang, Q.-Y.; Wu, L.-Y.; Wang, extent of current studies about the relationship between exosomal microRNAs and metastasis of Y.; Zhao, R.-C.; Xiong, L.-X.
    [Show full text]
  • Diffuse Large B-Cell Lymphoma with Combined TP53 Mutation and MIR34A Methylation: Another “Double Hit” Lymphoma with Very Poor Outcome?
    www.impactjournals.com/oncotarget/ Oncotarget, Vol. 5, No. 7 Diffuse large B-cell lymphoma with combined TP53 mutation and MIR34A methylation: Another “double hit” lymphoma with very poor outcome? Fazila Asmar1,*, Christoffer Hother1,*, Gorjan Kulosman1, Marianne Bach Treppendahl1, Helene Myrtue Nielsen1, Ulrik Ralfkiaer1,2, Anja Pedersen1, Michael Boe Møller3, Elisabeth Ralfkiaer2, Peter de Nully Brown1, and Kirsten Grønbæk1 1 Department of Hematology, Rigshospitalet, Copenhagen, Denmark, 2 Department of Pathology, Rigshospitalet, Copenhagen, Denmark, 3 Department of Pathology, Odense University Hospital, Odense, Denmark. * These authors contributed equally to the paper Correspondence to:Kirsten Grønbæk, email: [email protected] Keywords: Epigenetic changes, DNA methylation, microRNA, Tumor suppressors, non-Hodgkin lymphoma Received: February 9, 2014 Accepted: March 30, 2014 Published: March 31, 2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT: MiR34A, B and C have been implicated in lymphomagenesis, but information on their role in normal CD19+ B-cells (PBL-B) and de novo diffuse large B-cell lymphoma (DLBCL) is limited. We show that in normal and activated B-cells miR34A-5p plays a dominant role compared to other miR34 family members. Only miR34A-5p is expressed in PBL-B, and significantly induced in activated B-cells and reactive lymph nodes. In PBL-B, the MIR34A and MIR34B/C promoters are unmethylated, but the latter shows enrichment for the H3K4me3/H3K27me3 silencing mark. Nine de novo DLBCL cases (n=150) carry both TP53 mutation and MIR34A methylation (“double hit”) and these patients have an exceedingly poor prognosis with a median survival of 9.4 months (P<0.0001), while neither TP53 mutation, MIR34A or MIR34B/C promoter methylation alone (“single hit”) influence on survival.
    [Show full text]
  • Micrornas Tune Cerebral Cortical Neurogenesis
    Cell Death and Differentiation (2012) 19, 1573–1581 & 2012 Macmillan Publishers Limited All rights reserved 1350-9047/12 www.nature.com/cdd Review MicroRNAs tune cerebral cortical neurogenesis M-L Volvert1,2, F Rogister1,2, G Moonen1,2, B Malgrange1,2 and L Nguyen*,1,2,3 MicroRNAs (miRNAs) are non-coding RNAs that promote post-transcriptional silencing of genes involved in a wide range of developmental and pathological processes. It is estimated that most protein-coding genes harbor miRNA recognition sequences in their 30 untranslated region and are thus putative targets. While functions of miRNAs have been extensively characterized in various tissues, their multiple contributions to cerebral cortical development are just beginning to be unveiled. This review aims to outline the evidence collected to date demonstrating a role for miRNAs in cerebral corticogenesis with a particular emphasis on pathways that control the birth and maturation of functional excitatory projection neurons. Cell Death and Differentiation (2012) 19, 1573–1581; doi:10.1038/cdd.2012.96; published online 3 August 2012 Facts underlie cognitive, motor and perceptual abilities.1 Most cortical neurons are glutamatergic projection neurons that MicroRNAs are enriched in the nervous system and are born in germinal compartment of dorsal telencephalon. some show a dynamic expression that correlates with They migrate a short distance along radial glia fibers to settle important milestones of cerebral cortex development. in dedicated cortical layers. They send axonal projections to Mouse embryos harbor cortical malformations upon Dicer distant cortical or subcortical targets. The second class of deletion. neurons includes GABAergic interneurons that arise from the MicroRNAs underlie cortical development by ventral telencephalon and travel along multiple tangential repressing multiple target genes, some being intricately paths to reach the cortical wall.
    [Show full text]
  • Mir-34A Regulates Expression of the Stathmin-1 Oncoprotein and Prostate Cancer Progression Balabhadrapatruni V.S.K
    Published OnlineFirst October 12, 2017; DOI: 10.1158/1541-7786.MCR-17-0230 Oncogenes and Tumor Suppressors Molecular Cancer Research miR-34a Regulates Expression of the Stathmin-1 Oncoprotein and Prostate Cancer Progression Balabhadrapatruni V.S.K. Chakravarthi1,2, Darshan S. Chandrashekar1, Sumit Agarwal1, Sai Akshaya Hodigere Balasubramanya1, Satya S. Pathi3, Moloy T. Goswami3, Xiaojun Jing3,4, Rui Wang3, Rohit Mehra3,4,5, Irfan A. Asangani3, Arul M. Chinnaiyan3,4,5,6,7, Upender Manne1,2, Guru Sonpavde8, George J. Netto1, Jennifer Gordetsky1, and Sooryanarayana Varambally1,2,3 Abstract In aggressive prostate cancers, the oncoprotein STMN1 (also regulated expression of miR-34a. Furthermore, gene expression known as stathmin 1 and oncoprotein 18) is often overexpressed. profiling of STMN1-modulated prostate cancer cells revealed STMN1 is involved in various cellular processes, including cell molecular alterations, including elevated expression of growth proliferation, motility, and tumor metastasis. Here, it was found differentiation factor 15 (GDF15), which is involved in cancer that the expression of STMN1 RNA and protein is elevated in progression and potentially in STMN1-mediated oncogenesis. metastatic prostate cancers. Knockdown of STMN1 resulted in Thus, in prostate cancer, CtBP1-regulated miR-34a modulates reduced proliferation and invasion of cells and tumor growth and STMN1 expression and is involved in cancer progression through metastasis in vivo. Furthermore, miR-34a downregulated STMN1 the CtBP1\miR-34a\STMN1\GDF15 axis. by directly binding to its 30-UTR. Overexpression of miR-34a in prostate cancer cells reduced proliferation and colony formation, Implications: The CtBP1\miR-34a\STMN1\GDF15 axis is a suggesting that it is a tumor suppressor.
    [Show full text]
  • Emerging Evidence for Micrornas As Regulators of Cancer Stem Cells
    Cancers 2011, 3, 3957-3971; doi:10.3390/cancers3043957 OPEN ACCESS cancers ISSN 2072-6694 www.mdpi.com/journal/cancers Review Emerging Evidence for MicroRNAs as Regulators of Cancer Stem Cells Aisha Sethi 1 and Lynette M. Sholl 2,* 1 Department of Pathology, Henry Ford Hospital, Detroit, MI 48202, USA; E-Mail: [email protected] 2 Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA * Author to whom correspondence should be addressed: E-Mail: [email protected]; Tel.: +1-617-732-7510; Fax: +1-617-277-9015. Received: 25 August 2011; in revised form: 1 October 2011 / Accepted: 13 October 2011 / Published: 24 October 2011 Abstract: Cancer stem cells are defined as a subpopulation of cells within a tumor that are capable of self-renewal and differentiation into the heterogeneous cell lineages that comprise the tumor. Many studies indicate that cancer stem cells may be responsible for treatment failure and relapse in cancer patients. The factors that regulate cancer stem cells are not well defined. MicroRNAs (miRNAs) are small non-coding RNAs that regulate translational repression and transcript degradation. miRNAs play a critical role in embryonic and inducible pluripotent stem cell regulation and emerging evidence supports their role in cancer stem cell evolution. To date, miRNAs have been shown to act either as tumor suppressor genes or oncogenes in driving critical gene expression pathways in cancer stem cells in a wide range of human malignancies, including hematopoietic and epithelial tumors and sarcomas. miRNAs involved in cancer stem cell regulation provide attractive, novel therapeutic targets for cancer treatment.
    [Show full text]
  • An Exportin-1–Dependent Microrna Biogenesis Pathway During Human Cell Quiescence
    An Exportin-1–dependent microRNA biogenesis PNAS PLUS pathway during human cell quiescence Ivan Martineza,1,2, Karen E. Hayesa,1, Jamie A. Barra, Abby D. Harolda, Mingyi Xieb, Syed I. A. Bukharic, Shobha Vasudevanc, Joan A. Steitzd,e,f,2, and Daniel DiMaiod,f,g,h aDepartment of Microbiology, West Virginia University Cancer Institute, West Virginia University, Morgantown, WV 26506; bDepartment of Biochemistry and Molecular Biology, University of Florida Health Cancer Center, University of Florida, Gainesville, FL 32610; cCancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114; dDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06536; eHoward Hughes Medical Institute, Yale University, New Haven, CT 06536; fYale Cancer Center, New Haven, CT 06520; gDepartment of Genetics, Yale School of Medicine, New Haven, CT 06510; and hDepartment of Therapeutic Radiology, Yale School of Medicine, New Haven, CT 06510 Contributed by Joan A. Steitz, May 11, 2017 (sent for review November 14, 2016; reviewed by Judith Campisi, Richard I. Gregory, and Narry Kim) The reversible state of proliferative arrest known as “cellular qui- mature miRNA. Finally, the guide miRNA strand is loaded into escence” plays an important role in tissue homeostasis and stem the RNA-induced silencing complex (RISC) containing one of cell biology. By analyzing the expression of miRNAs and miRNA- four Argonaute proteins and GW182 protein at its core (18–20). processing factors during quiescence in primary human fibroblasts, Recent studies have identified alternative pathways of miRNA we identified a group of miRNAs that are induced during quies- biogenesis in different cell and animal models: pre-miRNA/intron cence despite markedly reduced expression of Exportin-5, a pro- miRNAs (miRtrons), which are Drosha and DGCR8 indepen- tein required for canonical miRNA biogenesis.
    [Show full text]