Abstract 13123: Noncoding RNAs in Early Mouse Embryonic Stem Cell Commitment to Cardiovascular Precursors
Introduction: It has been demonstrated that nitric oxide (NO) induces chromatin remodeling and, in mouse embryonic stem cells (mES), it is responsible for a decreased expression of stemness-associated genes including Oct4, Nanog, Sox2 and KLF4. During this process, NO negatively regulates the zinc-finger E-box binding homeobox 1 and 2 (ZEBs) transcription repression factors via miR-200 family members.
Hypothesis: Objective of the present work is to understand the molecular mechanism by which NO, ZEB factors and miR-200 family cooperated to maintain stemness in mES.
Methods and Results: In these experiments mES were cultured in 3 different conditions: 1) stem condition, achieved by “ground state” medium (DMEM + 10% FBS + 3 inhibitors: leukemia inhibitory factor (LIF); ERK inhibitor; and GSK3 inhibitor); 2) spontaneous differentiation (DMEM + 10% FBS); 3) NO-dependent mesendodermal differentiation (DMEM + 10% FBS + 250 μM NO donor DETA/NO). In the presence of NO, the combination of RNA-Seq experiments by next generation sequencing as well as targeted real time PCR expression analyses showed the presence of lineage specific mesendodermal markers and concurrent down modulation of neuroectodermal markers in mES. In addition to the up regulation of the miR-200 family that inhibited expression of mesendodermal markers, this analysis identified 4 long non-coding RNAs (LncRNAs) highly expressed in undifferentiated mES and 6 different LncRNAs up-regulated after inhibition withdrawal. One of the candidates, the LncRNA Gtl2-as, which has been recently reported to interact with the Polycomb complex, was significantly increased after exposure to NO. Moreover, a RNA-protein interaction prediction analysis identified potential binding elements for ZEB factors, suggesting a new molecular regulation pathway for control of stem cell function.
Conclusions: Our data provide the first indication of a molecular interplay between factors ZEB1 and 2 and selected LncRNAs during mES stemness maintenance and early commitment to the mesoderm.
Author Disclosures: C. Cencioni: None. M. Savoia: None. F. Spallotta: None. C. Kuenne: None. S. Guenther: None. A.M. Zeiher: None. C. Gaetano: None.
- © 2014 by American Heart Association, Inc.