title: miR-15b/16 protects primary human retinal microvascular endothelial cells against hyperglycemia-induced increases in tumor necrosis factor alpha and suppressor of cytokine signaling 3.
abstract: BACKGROUND
  Mechanisms underlying the pathology of diabetic retinopathy are still not completely understood. Increased understanding of potential cellular pathways responsive to hyperglycemia is essential to develop novel therapeutic strategies for diabetic retinopathy. Emerging evidence shows the impact of microRNA (miR) as a potential novel therapeutic target. The purpose of our study was to test the hypothesis that miR-15b and miR-16 are altered by hyperglycemia in retinal endothelial cells (REC), and that miR-15b/16 play key roles in regulating insulin signaling through a reduction in TNFα- and suppressor of cytokine signaling 3 (SOCS3)-mediated insulin resistance pathways.
  
  
  METHODS
  Human REC were maintained in normal (5 mM) glucose or transferred to high-glucose medium (25 mM) for 3 days. REC were transfected with miRNA mimics (hsa-miR-15b-5p and hsa-miR-16-5p) 48 h before cell harvest. A final concentration of 30 nM was used when transfected separately (miR-15b and miR-16) and 15 nM was used in combination (miR-15b + miR-16). A negative control group was treated with an equal concentration of a mimic negative control. The levels of miRNA overexpression were verified using quantitative reverse transcription-polymerase chain reaction and real-time PCR. Western blot analyses were performed to study the levels of phosphorylated Akt (Serine 473), Akt, SOCS3, insulin receptor, phosphorylated insulin receptor (tyrosine 1150/1151), and insulin receptor phosphorylated on Tyr960. In addition, ELISA was used to examine cleaved caspase 3 and TNFα. Analyses were done using unpaired Student t test. Data are presented as mean ± S.E.M.
  
  
  RESULTS
  We demonstrated that the expression of miR-15b and miR-16 was reduced in human REC cultured in hyperglycemia. Overexpression of miR-15b and/or miR-16 reduced TNFα and SOCS3 levels, while increasing insulin-like growth factor binding protein-3 (IGFBP-3) levels and the phosphorylation of insulin receptor (IR)(Tyr1150/1151) in REC cultured in hyperglycemia. These, in turn, led to an increase of Akt phosphorylation and decreased cleavage of caspase 3.
  
  
  CONCLUSIONS
  miR-15b and miR-16 play a role in the inhibition of insulin resistance via reduced TNFα and SOCS3 signaling and increased IGFBP-3 levels, resulting in REC protection from hyperglycemia-induced apoptosis. This outcome suggests that both miR-15b and miR-16 are potential therapeutic targets for therapeutics for the diabetic retina.
authors.affiliation: Department of Anatomy and Cell Biology, Wayne State University, 9314 Scott Hall, 48201, Detroit, MI, USA. eunye@med.wayne.edu.
authors.forename: Eun-Ah
authors.initials: EA
authors.lastname: Ye
authors.affiliation: Department of Anatomy and Cell Biology, Wayne State University, 9314 Scott Hall, 48201, Detroit, MI, USA. jsteinle@med.wayne.edu.|Department of Ophthalmology, Wayne State University, 9314 Scott Hall, 48201, Detroit, MI, USA. jsteinle@med.wayne.edu.
authors.forename: Jena J
authors.initials: JJ
authors.lastname: Steinle
mesh_terms: D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects*; D005947:Glucose / Q000633:toxicity*; D006801:Humans; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics / Q000378:metabolism; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000166:cytology*; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D016207:Cytokines; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D018972:Insulin-Like Growth Factor Binding Protein 3; C505361:MIRN15 microRNA, human; C505362:MIRN16 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; C106644:SOCS3 protein, human; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D014409:Tumor Necrosis Factor-alpha; D053148:Caspase 3; D005947:Glucose