title: MicroRNA miR-466 inhibits Lymphangiogenesis by targeting prospero-related homeobox 1 in the alkali burn corneal injury model.
abstract: BACKGROUND
  Lymphangiogenesis is one of the major causes of corneal graft rejection. Among the lymphangiogenic factors, vascular endothelial growth factor (VEGF)-C and -D are considered to be the most potent. Both bind to VEGF receptor 3 (VEGFR3) to activate Prospero homeobox 1 (Prox1), a transcription factor essential for the development and maintenance of lymphatic vasculature. MicroRNAs (miRNAs) bind to the 3' untranslated regions (3' UTRs) of target genes in a sequence-specific manner and suppress gene expression. In the current study, we searched for miRNAs that target the pro-lymphangiogenic factor Prox1.
  
  
  RESULTS
  Among the miRNAs predicted by the bioinformatic analysis to seed match with the 3' UTR of Prox-1, we chose 3 (miR-466, miR-4305, and miR-4795-5p) for further investigation. Both the miR-466 and miR-4305 mimics, but not the miR-4795-5p mimic, significantly reduced the luciferase activity of the Prox-1 3' UTR reporter vector. In primary lymphatic endothelial cells (HDLEC), miR-466 mimic transfection suppressed Prox1 mRNA and protein expression, while miR-4305 mimic transfection did not. Experiments using mutated reporter constructs of the two possible seed match sites on the 3' UTR of Prox1 suggested that the target site 2 directly bound miR-466. HDLEC transfected with the miR-466 mimic suppressed tube formation as compared to the scrambled control. Furthermore, HDLEC transfected with a miR-466 inhibitor showed enhanced tube formation as compared to control inhibitor transfected cells, and this inhibitory effect was counteracted by Prox1 siRNA. The miR-466 mimic reduced angiogenesis and lymphangiogenesis resulting in clearer corneas in an cornea injury rat model compared to the scrambled control.
  
  
  CONCLUSIONS
  Our data suggest that miR-446 may have a protective effect on transplanted corneas by suppressing Prox1 expression at the post-transcriptional level. The results of the current study may provide insights into the mechanisms of lymphangiogenesis resulting from corneal graft rejection and alkali-burn injuries, as well as into the development of new treatments for lymphangiogenic eye diseases.
authors.affiliation: Department of Medical Lifescience, College of Medicine, The Catholic University of Korea, Seoul, Korea. ongiaskor@gmail.com.
authors.forename: Minkoo
authors.initials: M
authors.lastname: Seo
authors.affiliation: Catholic Institute for Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea. junschoi@catholic.ac.kr.
authors.forename: Jun-Sub
authors.initials: JS
authors.lastname: Choi
authors.affiliation: Catholic Institute for Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea. menard@catholic.ac.kr.|Department of Ophthalmology and Visual Science, Daejeon St. Mary's Hospital, Daejeon, Korea. menard@catholic.ac.kr.
authors.forename: Chang Rae
authors.initials: CR
authors.lastname: Rho
authors.affiliation: Catholic Institute for Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea. ckjoo@catholic.ac.kr.|Department of Ophthalmology and Visual Science, Seoul St. Mary's Hospital, Seoul, Korea. ckjoo@catholic.ac.kr.
authors.forename: Choun-Ki
authors.initials: CK
authors.lastname: Joo
authors.affiliation: Department of Medical Lifescience, College of Medicine, The Catholic University of Korea, Seoul, Korea. sukklee@catholic.ac.kr.
authors.forename: Suk Kyeong
authors.initials: SK
authors.lastname: Lee
mesh_terms: D000468:Alkalies / Q000633:toxicity; D000818:Animals; D002057:Burns, Chemical / Q000209:etiology / Q000235:genetics*; D065306:Corneal Injuries / Q000139:chemically induced / Q000235:genetics*; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism; D005126:Eye Burns / Q000139:chemically induced / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D042583:Lymphangiogenesis; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism; D000098551:Prospero-Related Homeobox 1 Protein
chemical_list: D000468:Alkalies; D018398:Homeodomain Proteins; C585904:MIRN466 microRNA, human; D035683:MicroRNAs; D025521:Tumor Suppressor Proteins; D000098551:Prospero-Related Homeobox 1 Protein