BackgroundLymphangiogenesis 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.ResultsAmong 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-014-0104-0) contains supplementary material, which is available to authorized users.

Choosing the corneal incision based on the preoperative steep meridian significantly decreased keratometric astigmatism at the temporal, superotemporal, and superior locations. Thus, it is desirable to place the corneal incision on the steep meridian in eyes with corneal astigmatism higher than 0.50 D.