title: MiR-9 regulates the post-transcriptional level of VEGF165a by targeting SRPK-1 in ARPE-19 cells.
abstract: OBJECTIVE
  To investigate the effect of the overexpression of miRNA-9 to the ratio of pro- and anti-angiogenic isoforms of vascular endothelial growth factor (VEGF) in human retinal pigment cells (ARPE-19).
  
  
  METHODS
  Oxidative stress was induced to ARPE-19 cells by 4-hydroxynonenal (4-HNE), tert-butyl hydroperoxide (t-BH), and hypoxia chamber with 1% O₂. Expression patterns of miRNAs were validated by qPCR. Relative mRNA levels of VEGF and PEDF were measured by semi-quantitative PCR. After the transfection of miR-9 mimic and inhibitor, transcriptional levels of VEGF165a, VEGF 165b, and SRPK-1 were measured by qPCR.
  
  
  RESULTS
  We demonstrated that miR-9 expression is decreased in ARPE-19 human retinal pigment cells under hypoxic stress induced by 4-HNE, a lipid peroxidation end-product. We observed that miR-9 mimic transfection of ARPE-19 inhibited one of its targets, serine-arginine protein kinase-1 (SRPK-1), modulating the transcriptional level of VEGF165b. Transfection of miR-9 reduced the alternative splicing of VEGF165a mRNA in ARPE-19 cells under hypoxic conditions, suggesting that miR-mediated regulation of alternative splicing could be a potential therapeutic target in neovascular pathologies.
  
  
  CONCLUSIONS
  Hypoxic stress decreased the miR-9 level in ARPE-19 cells, which increased the transcriptional level of SRPK-1, resulting in alternative splicing shift to pro-angiogenic isoforms of VEGF165 in human retinal pigment epithelial cells.
authors.affiliation: Department of Anatomy, College of Medicine, Inje University, Bokji-ro 75, Busanjin-gu, Busan, South Korea, 614-735.
authors.forename: Changshin
authors.initials: C
authors.lastname: Yoon
authors.forename: Daejin
authors.initials: D
authors.lastname: Kim
authors.forename: Seonghan
authors.initials: S
authors.lastname: Kim
authors.forename: Ga Bin
authors.initials: GB
authors.lastname: Park
authors.forename: Dae Young
authors.initials: DY
authors.lastname: Hur
authors.forename: Jae Wook
authors.initials: JW
authors.lastname: Yang
authors.forename: Sae Gwang
authors.initials: SG
authors.lastname: Park
authors.forename: Yeong Seok
authors.initials: YS
authors.lastname: Kim
mesh_terms: D000447:Aldehydes / Q000633:toxicity; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005136:Eye Proteins / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D035683:MicroRNAs / Q000235:genetics*; D009414:Nerve Growth Factors / Q000235:genetics; D018384:Oxidative Stress; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism*; D015843:Serpins / Q000235:genetics; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*; D020122:tert-Butylhydroperoxide / Q000633:toxicity; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D000447:Aldehydes; D005136:Eye Proteins; D035683:MicroRNAs; D009414:Nerve Growth Factors; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D015843:Serpins; D042461:Vascular Endothelial Growth Factor A; D020122:tert-Butylhydroperoxide; D000099174:Pigment Epithelium-Derived Factor; C541874:MIRN92 microRNA, human; C467484:VEGFA protein, human; C087728:SRPK1 protein, human; C027576:4-hydroxy-2-nonenal