title: Expression and cellular localization of microRNA-29b and RAX, an activator of the RNA-dependent protein kinase (PKR), in the retina of streptozotocin-induced diabetic rats.
abstract: OBJECTIVE
  The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats.
  
  
  METHODS
  Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis.
  
  
  RESULTS
  We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b.
  
  
  CONCLUSIONS
  Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.
authors: Department of Biochemistry and Immunology, School of Medicine University of São Paulo, Ribeirão Preto, Brazil.
authors: Viviane A O
authors: VA
authors: Silva
authors: Anna
authors: A
authors: Polesskaya
authors: Thaís A
authors: TA
authors: Sousa
authors: Vani M A
authors: VM
authors: Corrêa
authors: Nayara Delgado
authors: ND
authors: André
authors: Rosana I
authors: RI
authors: Reis
authors: Isis C
authors: IC
authors: Kettelhut
authors: Annick
authors: A
authors: Harel-Bellan
authors: Fernando L
authors: FL
authors: De Lucca
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D003921:Diabetes Mellitus, Experimental / Q000235:genetics / Q000378:metabolism*; D003930:Diabetic Retinopathy / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D017403:In Situ Hybridization; D008156:Luciferases / Q000032:analysis; D008297:Male; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000378:metabolism*; D051248:Retinal Horizontal Cells / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics*; D015854:Up-Regulation; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
chemical_list: D018398:Homeodomain Proteins; C526366:MIRN29 microRNA, rat; D035683:MicroRNAs; C512581:RAX protein, rat; D008156:Luciferases; D019892:eIF-2 Kinase