Genome‐Wide Analysis of miRNA‐mRNA Interactions in Marrow Stromal Cells

Regulation of hematopoietic stem cell proliferation, lineage commitment, and differentiation in adult vertebrates requires extrinsic signals provided by cells in the marrow microenvironment (ME) located within the bone marrow. Both secreted and cell‐surface bound factors critical to this regulation have been identified, yet control of their expression by cells within the ME has not been addressed. Herein we hypothesize that microRNAs (miRNAs) contribute to their controlled expression. MiRNAs are small noncoding RNAs that bind to target mRNAs and downregulate gene expression by either initiating mRNA degradation or preventing peptide translation. Testing the role of miRNAs in downregulating gene expression has been difficult since conventional techniques used to define miRNA‐mRNA interactions are indirect and have high false‐positive and negative rates. In this report, a genome‐wide biochemical technique (high‐throughput sequencing of RNA isolated by cross‐linking immunoprecipitation or HITS‐CLIP) was used to generate unbiased genome‐wide maps of miRNA‐mRNA interactions in two critical cellular components of the marrow ME: marrow stromal cells and bone marrow endothelial cells. Analysis of these datasets identified miRNAs as direct regulators of JAG1, WNT5A, MMP2, and VEGFA; four factors that are important to ME function. Our results show the feasibility and utility of unbiased genome‐wide biochemical techniques in dissecting the role of miRNAs in regulation of complex tissues such as the marrow ME. Stem Cells 2014;32:662–673

[1]  C. Shin,et al.  Annals of the New York Academy of Sciences Microrna–target Interactions: New Insights from Genome-wide Approaches , 2022 .

[2]  N. Ferrara,et al.  VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism , 2002, Nature.

[3]  B. Trask,et al.  The human homolog of rat Jagged1 expressed by marrow stroma inhibits differentiation of 32D cells through interaction with Notch1. , 1998, Immunity.

[4]  A. Trumpp,et al.  Bone-marrow haematopoietic-stem-cell niches , 2006, Nature Reviews Immunology.

[5]  B. Sacchetti,et al.  Self-Renewing Osteoprogenitors in Bone Marrow Sinusoids Can Organize a Hematopoietic Microenvironment , 2007, Cell.

[6]  P. Zamore,et al.  Small silencing RNAs: an expanding universe , 2009, Nature Reviews Genetics.

[7]  G. Opdenakker,et al.  Zymography methods for visualizing hydrolytic enzymes , 2013, Nature Methods.

[8]  D. Phinney.  Functional heterogeneity of mesenchymal stem cells: Implications for cell therapy , 2012, Journal of cellular biochemistry.

[9]  Jernej Ule,et al.  CLIP: a method for identifying protein-RNA interaction sites in living cells. , 2005, Methods.

[10]  L. Gibson,et al.  MMP-2 is required for bone marrow stromal cell support of pro-B-cell chemotaxis. , 2005, Experimental hematology.

[11]  H. Clevers.  Wnt/beta-catenin signaling in development and disease. , 2006, Cell.

[12]  P. Schedin,et al.  Reproductive state of rat mammary gland stroma modulates human breast cancer cell migration and invasion. , 2000, Cancer research.

[13]  P. Bianco.  Bone and the hematopoietic niche: a tale of two stem cells. , 2011, Blood.

[14]  F. D. de Sauvage,et al.  Hematopoietic Deficiencies in c‐mpl and TPO Knockout Mice , 1998, Stem cells.

[15]  N. Rajewsky.  microRNA target predictions in animals , 2006, Nature Genetics.

[16]  D. Bodine,et al.  Wnt5a inhibits canonical Wnt signaling in hematopoietic stem cells and enhances repopulation , 2007, Proceedings of the National Academy of Sciences.

[17]  Hans Clevers,et al.  Wnt/β-Catenin Signaling in Development and Disease , 2006, Cell.

[18]  P. Sonneveld,et al.  Multiple Human Vault RNAs , 2001, The Journal of Biological Chemistry.

[19]  E. Ullian,et al.  A role for noncanonical microRNAs in the mammalian brain revealed by phenotypic differences in Dgcr8 versus Dicer1 knockouts and small RNA sequencing. , 2011, RNA.

[20]  Ana Kozomara,et al.  miRBase: integrating microRNA annotation and deep-sequencing data , 2010, Nucleic Acids Res..

[21]  Charles P. Lin,et al.  Bone progenitor dysfunction induces myelodysplasia and secondary leukemia , 2010, Nature.

[22]  T. Nagasawa,et al.  Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches. , 2006, Immunity.

[23]  M. Zavolan,et al.  A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins , 2011, Nature Methods.

[24]  Ryan T Fuchs,et al.  Structural bias in T4 RNA ligase-mediated 3′-adapter ligation , 2012, Nucleic acids research.

[25]  D. Scadden,et al.  Stem-cell ecology and stem cells in motion. , 2008, Blood.

[26]  A. T. Freitas,et al.  Current tools for the identification of miRNA genes and their targets , 2009, Nucleic acids research.

[27]  Yong Sun Lee,et al.  Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity. , 2011, RNA.

[28]  J. Barker.  Sl/Sld hematopoietic progenitors are deficient in situ. , 1994, Experimental hematology.

[29]  Gene W. Yeo,et al.  An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells , 2009, Nature Structural &Molecular Biology.

[30]  Robert B Darnell,et al.  HITS‐CLIP: panoramic views of protein–RNA regulation in living cells , 2010, Wiley interdisciplinary reviews. RNA.

[31]  A. Mele,et al.  Ago HITS-CLIP decodes miRNA-mRNA interaction maps , 2009, Nature.

[32]  Scott B. Dewell,et al.  Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP , 2010, Cell.

[33]  Y. Pekarsky,et al.  Reprogramming of miRNA networks in cancer and leukemia. , 2010, Genome research.

[34]  Michel Vaubourdolle,et al.  A protocol for isolation and culture of human umbilical vein endothelial cells , 2007, Nature Protocols.

[35]  Daniel Herschlag,et al.  Systematic Identification of mRNAs Recruited to Argonaute 2 by Specific microRNAs and Corresponding Changes in Transcript Abundance , 2008, PloS one.

[36]  T. Suda,et al.  Hematopoietic stem cells and their niche. , 2005, Trends in immunology.

[37]  N. Rajewsky,et al.  A human snoRNA with microRNA-like functions. , 2008, Molecular cell.

[38]  K. Tokoyoda,et al.  Long-term hematopoietic stem cells require stromal cell-derived factor-1 for colonizing bone marrow during ontogeny. , 2003, Immunity.

[39]  T. Austin,et al.  A role for the Wnt gene family in hematopoiesis: expansion of multilineage progenitor cells. , 1997, Blood.

[40]  R. Alon,et al.  The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow. , 1999, The Journal of clinical investigation.

[41]  J. Griffin,et al.  Notch receptors and hematopoiesis. , 2001, Experimental hematology.

[42]  B. Torok-Storb,et al.  Functionally distinct human marrow stromal cell lines immortalized by transduction with the human papilloma virus E6/E7 genes. , 1995, Blood.

[43]  B. Weksler,et al.  Characterization of a newly established human bone marrow endothelial cell line: distinct adhesive properties for hematopoietic progenitors compared with human umbilical vein endothelial cells. , 1997, Laboratory investigation; a journal of technical methods and pathology.

[44]  M. Gerstein,et al.  RNA-Seq: a revolutionary tool for transcriptomics , 2009, Nature Reviews Genetics.

[45]  David Tollervey,et al.  Cross-linking, ligation, and sequencing of hybrids reveals RNA–RNA interactions in yeast , 2011, Proceedings of the National Academy of Sciences.

[46]  Ben D. MacArthur,et al.  Mesenchymal and haematopoietic stem cells form a unique bone marrow niche , 2010, Nature.

[47]  T. Suda,et al.  Isolation and characterization of endosteal niche cell populations that regulate hematopoietic stem cells. , 2010, Blood.

[48]  Gary D. Bader,et al.  Cytoscape Web: an interactive web-based network browser , 2010, Bioinform..

[49]  杉山 立樹.  Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches , 2007 .

[50]  R. Taichman,et al.  G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4 , 2002, Nature Immunology.

[51]  Sonja J. Prohaska,et al.  Evolution of vault RNAs. , 2009, Molecular biology and evolution.

[52]  Rongkun Shen,et al.  Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach , 2012, Proceedings of the National Academy of Sciences.

[53]  Marian Thomson,et al.  Analysis of the genome sequences of three Drosophila melanogaster spontaneous mutation accumulation lines. , 2009, Genome research.

[54]  Lei Ding,et al.  Endothelial and perivascular cells maintain haematopoietic stem cells , 2011, Nature.

[55]  C. Bracken,et al.  Experimental strategies for microRNA target identification , 2011, Nucleic acids research.

[56]  P. Rakic,et al.  Processing of the notch ligand delta by the metalloprotease Kuzbanian. , 1999, Science.

[57]  Keisuke Ito,et al.  Tie2/Angiopoietin-1 Signaling Regulates Hematopoietic Stem Cell Quiescence in the Bone Marrow Niche , 2004, Cell.

[58]  A. Matsuzaki,et al.  The Hematopoietic Microenvironment: Matrix Metalloproteinases in the Hematopoietic Microenvironment. , 2000, Hematology.

[59]  L. Claesson‐Welsh,et al.  VEGF receptor signalling ? in control of vascular function , 2006, Nature Reviews Molecular Cell Biology.

[60]  A. Matsuzaki,et al.  Matrix Metalloproteinases in the Hematopoietic Microenvironment. , 1999, Hematology.

[61]  M. Ladomery,et al.  Alternative splicing in angiogenesis: the vascular endothelial growth factor paradigm. , 2007, Cancer letters.

[62]  Paul Zhang,et al.  A novel monoclonal antibody against human Argonaute proteins reveals unexpected characteristics of miRNAs in human blood cells. , 2007, RNA.

[63]  D. Bartel,et al.  MicroRNAs Modulate Hematopoietic Lineage Differentiation , 2004, Science.

[64]  Johan Vallon-Christersson,et al.  The non-coding RNA of the multidrug resistance-linked vault particle encodes multiple regulatory small RNAs , 2009, Nature Cell Biology.

[65]  Göran Karlsson,et al.  Signaling pathways governing stem-cell fate. , 2008, Blood.

[66]  S. Linnarsson,et al.  Counting absolute numbers of molecules using unique molecular identifiers , 2011, Nature Methods.

[67]  Xiaodong Yang,et al.  MiR-886-3p Down Regulates CXCL12 (SDF1) Expression in Human Marrow Stromal Cells , 2010, PloS one.

[68]  Á..  Functionally Distinct Human Marrow Stromal Cell Lines Immortalized by Transduction With the Human Papilloma Virus E 6 / E 7 Genes , 2002 .

[69]  Henry Yang,et al.  MicroRNA-125b Promotes Neuronal Differentiation in Human Cells by Repressing Multiple Targets , 2009, Molecular and Cellular Biology.

[70]  S. Karpatkin,et al.  Identification of a family of alternatively spliced mRNA species of angiopoietin-1. , 2000, Blood.

[71]  M. Landthaler,et al.  Transcriptome-wide analysis of protein-RNA interactions using high-throughput sequencing. , 2012, Seminars in cell & developmental biology.

[72]  J. Aster,et al.  Notch signaling in hematopoiesis and early lymphocyte development , 2002, Immunological reviews.

[73]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[74]  S. Morrison,et al.  Uncertainty in the niches that maintain haematopoietic stem cells , 2008, Nature Reviews Immunology.