title: MicroRNA-101 is repressed by EZH2 and its restoration inhibits tumorigenic features in embryonal rhabdomyosarcoma.
abstract: BACKGROUND
  Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma arising from myogenic precursors that have lost their capability to differentiate into skeletal muscle. The polycomb-group protein EZH2 is a Lys27 histone H3 methyltransferase that regulates the balance between cell proliferation and differentiation by epigenetically silencing muscle-specific genes. EZH2 is often over-expressed in several human cancers acting as an oncogene. We previously reported that EZH2 inhibition induces cell cycle arrest followed by myogenic differentiation of RMS cells of the embryonal subtype (eRMS). MiR-101 is a microRNA involved in a negative feedback circuit with EZH2 in different normal and tumor tissues. To that, miR-101 can behave as a tumor suppressor in several cancers by repressing EZH2 expression. We, therefore, evaluated whether miR-101 is de-regulated in eRMS and investigated its interplaying with EZH2 as well as its role in the in vitro tumorigenic potential of these tumor cells.
  
  
  RESULTS
  Herein, we report that miR-101 is down-regulated in eRMS patients and in tumor cell lines compared to their controls showing an inverse pattern of expression with EZH2. We also show that miR-101 is up-regulated in eRMS cells following both genetic and pharmacological inhibition of EZH2. In turn, miR-101 forced expression reduces EZH2 levels as well as restrains the migratory potential of eRMS cells and impairs their clonogenic and anchorage-independent growth capabilities. Finally, EZH2 recruitment to regulatory region of miR-101-2 gene decreases in EZH2-silenced eRMS cells. This phenomenon is associated to reduced H3K27me3 levels at the same regulatory locus, indicating that EZH2 directly targets miR-101 for repression in eRMS cells.
  
  
  CONCLUSIONS
  Altogether, our data show that, in human eRMS, miR-101 is involved in a negative feedback loop with EZH2, whose targeting has been previously shown to halt eRMS tumorigenicity. They also demonstrate that the re-induction of miR-101 hampers the tumor features of eRMS cells. In this scenario, epigenetic dysregulations confirm their crucial role in the pathogenesis of this soft tissue sarcoma.
authors.affiliation: Department of Oncohematology, Laboratory of Angiogenesis, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Vella
authors.affiliation: Laboratorio di Patologia Vascolare, Istituto Dermopatico dell'Immacolata, IRCCS, Rome, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Pomella
authors.affiliation: Department of Oncohematology, Laboratory of Angiogenesis, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy.
authors.forename: Pier Paolo
authors.initials: PP
authors.lastname: Leoncini
authors.affiliation: Department of Oncohematology, Laboratory of Angiogenesis, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Colletti
authors.affiliation: Department of Oncohematology, Laboratory of Angiogenesis, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Conti
authors.affiliation: Chemical Biology Laboratory, Frederick National Laboratory for Cancer Research, CCR, National Cancer Institute, NIH, Frederick, MD USA.
authors.forename: Victor E
authors.initials: VE
authors.lastname: Marquez
authors.affiliation: Department of Biological, Geological and Environmental Sciences, Biology Unit, University of Bologna, Bologna, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Strillacci
authors.affiliation: Laboratory of Translational Research in Paediatric Cancer, Vall d'Hebron Research Institute and Vall d'Hebron Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain.
authors.forename: Josep
authors.initials: J
authors.lastname: Roma
authors.affiliation: Laboratory of Translational Research in Paediatric Cancer, Vall d'Hebron Research Institute and Vall d'Hebron Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain.
authors.forename: Soledad
authors.initials: S
authors.lastname: Gallego
authors.affiliation: Department of Oncohematology, Clinical Unit, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy.
authors.forename: Giuseppe M
authors.initials: GM
authors.lastname: Milano
authors.affiliation: Laboratorio di Patologia Vascolare, Istituto Dermopatico dell'Immacolata, IRCCS, Rome, Italy.
authors.forename: Maurizio C
authors.initials: MC
authors.lastname: Capogrossi
authors.affiliation: Department of Oncohematology, Clinical Unit, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy.
authors.forename: Alice
authors.initials: A
authors.lastname: Bertaina
authors.affiliation: Laboratorio di Patologia Vascolare, Istituto Dermopatico dell'Immacolata, IRCCS, Rome, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Ciarapica
authors.affiliation: Department of Oncohematology, Laboratory of Angiogenesis, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Rota
keywords: Anchorage-independent growth; Cell motility; Cell proliferation; Chromatin immunoprecipitation; EZH2; Histone methyltransferases; MiR-101; Polycomb proteins; Rhabdomyosarcoma