title: MiR-126 overexpression inhibits high glucose-induced migration and tube formation of rhesus macaque choroid-retinal endothelial cells by obstructing VEGFA and PIK3R2.
abstract: OBJECTIVE
  The aims of this study are to investigate the relative regulation between miR-126 and VEGF/PI3K/AKT signaling pathway in retinal vascular endothelial cells.
  
  
  METHODS
  Rhesus macaque choroid-retinal endothelial cell line (RF/6A) cells were cultured in high glucose to imitate the conditions occurring in DR. First, we detected the expression of miR-126, VEGFA and PIK3R2 in RF/6A cells on the condition of high glucose by q-PCR and western blot. Then, after addition of miR-126 mimics and miR-126 inhibitor, we investigated the function of miR-126 in RF/6A cells by scratch wound, Transwell migration and tube formation assays, and the effect of miR-126 on the expression of VEGFA, PIK3R2 and AKT. Moreover, bioinformatics analysis and luciferase array were used to confirm the direct or specific regulation of miR-126 to VEGFA or PIK3R2.
  
  
  RESULTS
  Here, first, we found that high glucose could induce the decrease of miR-126 and the increase of VEGFA and PIK3R2 in RF/6A. Then, by scratch wound, Transwell migration and tube formation assays, we found that miR-126 overexpression could inhibit the migration and sprouting of RF/6A cells induced by high glucose, while knockdown of miR-126 led to the opposite results. Moreover, overexpression of miR-126 inhibited the increased expression of VEGFA, PIK3R2, SDF-1α, VCAM-1, and SPRED1, and the activation of AKT1 induced by high glucose and miR-126 inhibitor caused the opposite results which were determined by q-PCR and western blot. In addition, by luciferase assay, we found that miR-126 could directly negatively regulate VEGFA and PIK3R2.
  
  
  CONCLUSIONS
  Our results suggest that miR-126 overexpression inhibits the migration and sprouting of RF/6A cells induced by high glucose which might possibly be by blocking VEGFA and PIK3R2 in the VEGF/PI3K/AKT signaling pathway.
authors.affiliation: Department of Ophthalmology, Fourth Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650021, China.
authors.forename: Wen-Zhi
authors.initials: WZ
authors.lastname: Yang
authors.affiliation: Department of Osteology, First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Ophthalmology, Fourth Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650021, China.
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Xue
authors.affiliation: Department of Ophthalmology, Fourth Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650021, China.
authors.forename: Li-Bo
authors.initials: LB
authors.lastname: Xiao
authors.affiliation: Department of Ophthalmology, First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, China. Electronic address: liyanr@hotmail.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
mesh_terms: D000818:Animals; D002460:Cell Line; D002465:Cell Movement; D002829:Choroid / Q000378:metabolism* / Q000473:pathology; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019295:Computational Biology; D003930:Diabetic Retinopathy / Q000378:metabolism / Q000473:pathology; D005136:Eye Proteins / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D005947:Glucose / Q000378:metabolism*; D006943:Hyperglycemia / Q000378:metabolism / Q000473:pathology; D008253:Macaca mulatta; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D018716:Molecular Mimicry; D009994:Osmolar Concentration; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012313:RNA / Q000378:metabolism; D012160:Retina / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction; D013237:Stereoisomerism; D042461:Vascular Endothelial Growth Factor A / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D005136:Eye Proteins; D035683:MicroRNAs; D000081082:Phosphoinositide-3 Kinase Inhibitors; C039919:RNA, recombinant; D042461:Vascular Endothelial Growth Factor A; D012313:RNA; D058543:Class Ia Phosphatidylinositol 3-Kinase; D005947:Glucose
keywords: Diabetes mellitus; Diabetic retinopathy; PIK3R2; VEGFA; miR-126