title: Role of morphine, miR-212/132 and mu opioid receptor in the regulation of Bdnf in zebrafish embryos.
abstract: BACKGROUND
  Morphine is one of the first-line therapies for the treatment of pain despite its secondary effects. It modifies the expression of epigenetic factors like miRNAs. In the present study, we analyzed miR-212 and miR-132 and their implication in morphine effects in the zebrafish Central Nervous System (CNS) through the regulation of Bdnf expression.
  
  
  METHODS
  We used control and knock-down zebrafish embryos to assess the effects of morphine in miRNAs 212/132 and mitotic or apoptotic cells by qPCR, immunohistochemistry and TUNEL assay, respectively. Bdnf and TrkB were studied by western blot and through a primary neuron culture. A luciferase assay was performed to confirm the binding of miRNAs 212/132 to mecp2.
  
  
  RESULTS
  Morphine exposure decreases miR-212 but upregulates miR-132, as wells as Bdnf and TrkB, and changes the localization of proliferative cells. However, Bdnf expression was downregulated when miRNAs 212/132 and oprm1 were knocked-down. Furthermore, we proved that these miRNAs inhibit mecp2 expression by binding to its mRNA sequence. The described effects were corroborated in a primary neuron culture from zebrafish embryos.
  
  
  CONCLUSIONS
  We propose a mechanism in which morphine alters the levels of miRNAs 212/132 increasing Bdnf expression through mecp2 inhibition. oprm1 is also directly involved in this regulation. The present work confirms a relationship between the opioid system and neurotrophins and shows a key role of miR-212 and miR-132 on morphine effects through the regulation of Bdnf pathway.
  
  
  CONCLUSIONS
  miRNAs 212/132 are novel regulators of morphine effects on CNS. Oprm1 controls the normal expression of Bdnf.
authors.affiliation: Institute of Neurosciences of Castilla y Leon (INCyL), University of Salamanca, Spain; Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
authors.forename: Ada
authors.initials: A
authors.lastname: Jimenez-Gonzalez
authors.affiliation: Institute of Neurosciences of Castilla y Leon (INCyL), University of Salamanca, Spain; Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
authors.forename: Adrián
authors.initials: A
authors.lastname: García-Concejo
authors.affiliation: Institute of Neurosciences of Castilla y Leon (INCyL), University of Salamanca, Spain; Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
authors.forename: Saray
authors.initials: S
authors.lastname: López-Benito
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Salamanca, Spain; Institute of Neurosciences of Castilla y Leon (INCyL), University of Salamanca, Spain; Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.
authors.forename: Verónica
authors.initials: V
authors.lastname: Gonzalez-Nunez
authors.affiliation: Institute of Neurosciences of Castilla y Leon (INCyL), University of Salamanca, Spain; Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain; Department of cell biology and pathology, Salamanca, Spain.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Arévalo
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Salamanca, Spain; Institute of Neurosciences of Castilla y Leon (INCyL), University of Salamanca, Spain; Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain. Electronic address: requelmi@usal.es.
authors.forename: Raquel E
authors.initials: RE
authors.lastname: Rodriguez
mesh_terms: D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000032:analysis*; D018507:Gene Expression Regulation, Developmental; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000502:physiology*; D009020:Morphine / Q000494:pharmacology*; D020813:Receptor, trkB / Q000032:analysis; D017450:Receptors, Opioid, mu / Q000502:physiology*; D015027:Zebrafish / Q000196:embryology*
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D035683:MicroRNAs; D017450:Receptors, Opioid, mu; C000592936:mirn132 microRNA, zebrafish; C000609288:mirn212 microRNA, zebrafish; D009020:Morphine; D020813:Receptor, trkB
keywords: Bdnf; Danio rerio; Morphine; miR-132; miR-212