title: MIR221/MIR222-driven post-transcriptional regulation of P27KIP1 and P57KIP2 is crucial for high-glucose- and AGE-mediated vascular cell damage.
abstract: OBJECTIVE
  MicroRNAs (miRNAs) are a novel group of small non-coding RNAs that regulate gene expression at the post-transcriptional level and act on their target mRNAs in a tissue- and cell-type-specific manner. Herein, the relevance of MIR221/MIR222 in high-glucose- and AGE-mediated vascular damage was investigated.
  
  
  METHODS
  Functional studies were performed using human mature endothelial cells and endothelial progenitor cells subjected to high glucose or AGE. Quantitative real-time amplification was performed to analyse MIR221/MIR222 expression in these experimental conditions. Luciferase assay was used to identify MIR221/MIR222 targets. Functional studies were performed in vitro and in vivo in mice using gain- and loss-of-function approaches.
  
  
  RESULTS
  Using an in vivo mouse model we demonstrated that exposure to AGE and high glucose impaired vessel formation. Moreover, in vitro functional studies revealed that both high glucose and AGE inhibit cell-cycle progression by modulating the expression of P27KIP1 (also known as CDKN1B) and P57KIP2 (also known as CDKN1C), which encode cyclin-dependent kinase inhibitor 1B (p27, Kip1) (P27KIP1) and cyclin-dependent kinase inhibitor 1C (p57, Kip2) (P57KIP2), respectively. Crucial to AGE- and high-glucose-mediated cell-cycle arrest was the downregulation of MIR221/MIR222 expression. Luciferase assay showed that MIR221 and MIR222 specifically bind to the P27KIP1 and P57KIP2 mRNA 3'-untranslated regions, implicating P27KIP1 and P57KIP2 as MIR221/MIR222 targets. These results were confirmed by gain-of-function experiments in vitro, and by injecting mice with endothelial cells overexpressing MIR221 and MIR222.
  
  
  CONCLUSIONS
  We provide evidence that high-glucose- and AGE-induced inhibition of vascular cell proliferation is controlled by MIR221/MIR222-driven post-transcriptional regulation of P27KIP1 and P57KIP2. These data add further insight to the possible contribution of miRNAs in vascular damage mediated by a high-glucose environment.
authors: Department of Internal Medicine, University of Torino, Corso Dogliotti 14, 10126, Torino, Italy.
authors: G
authors: G
authors: Togliatto
authors: Department of Internal Medicine, University of Torino, Corso Dogliotti 14, 10126, Torino, Italy.
authors: A
authors: A
authors: Trombetta
authors: Department of Internal Medicine, University of Torino, Corso Dogliotti 14, 10126, Torino, Italy.
authors: P
authors: P
authors: Dentelli
authors: Department of Internal Medicine, University of Torino, Corso Dogliotti 14, 10126, Torino, Italy.
authors: A
authors: A
authors: Rosso
authors: Department of Internal Medicine, University of Torino, Corso Dogliotti 14, 10126, Torino, Italy. mariafelice.brizzi@unito.it.
authors: M F
authors: MF
authors: Brizzi
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism*; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D005947:Glucose / Q000494:pharmacology*; D017127:Glycation End Products, Advanced / Q000494:pharmacology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction
chemical_list: D050761:Cyclin-Dependent Kinase Inhibitor p57; D017127:Glycation End Products, Advanced; D035683:MicroRNAs; D050760:Cyclin-Dependent Kinase Inhibitor p27; D005947:Glucose