title: MicroRNA-34a modulates MDM4 expression via a target site in the open reading frame.
abstract: BACKGROUND
  MDM4, also called MDMX or HDMX in humans, is an important negative regulator of the p53 tumor suppressor. MDM4 is overexpressed in about 17% of all cancers and more frequently in some types, such as colon cancer or retinoblastoma. MDM4 is known to be post-translationally regulated by MDM2-mediated ubiquitination to decrease its protein levels in response to genotoxic stress, resulting in accumulation and activation of p53. At the transcriptional level, MDM4 gene regulation has been less clearly understood. We have reported that DNA damage triggers loss of MDM4 mRNA and a concurrent increase in p53 activity. These experiments attempt to determine a mechanism for down-regulation of MDM4 mRNA.
  
  
  RESULTS
  Here we report that MDM4 mRNA is a target of hsa-mir-34a (miR-34a). MDM4 mRNA contains a lengthy 3' untranslated region; however, we find that it is a miR-34a site within the open reading frame (ORF) of exon 11 that is responsible for the repression. Overexpression of miR-34a, but not a mutant miR-34a, is sufficient to decrease MDM4 mRNA levels to an extent identical to those of known miR-34a target genes. Likewise, MDM4 protein levels are decreased by miR-34a overexpression. Inhibition of endogenous miR-34a increased expression of miR-34a target genes and MDM4. A portion of MDM4 exon 11 containing this 8mer-A1 miR-34a site fused to a luciferase reporter gene is sufficient to confer responsiveness, being inhibited by additional expression of exogenous mir-34a and activated by inhibition of miR-34a.
  
  
  CONCLUSIONS
  These data establish a mechanism for the observed DNA damage-induced negative regulation of MDM4 and potentially provide a novel means to manipulate MDM4 expression without introducing DNA damage.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wright State University, Dayton, Ohio, United States of America.
authors.forename: Pooja
authors.initials: P
authors.lastname: Mandke
authors.forename: Nicholas
authors.initials: N
authors.lastname: Wyatt
authors.forename: Jillian
authors.initials: J
authors.lastname: Fraser
authors.forename: Benjamin
authors.initials: B
authors.lastname: Bates
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Berberich
authors.forename: Michael P
authors.initials: MP
authors.lastname: Markey
mesh_terms: D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics; D016366:Open Reading Frames; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D018797:Cell Cycle Proteins; C482775:MDM4 protein, human; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53