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Jongkook Park, Olga V Volpert, Robert M Lavker; microRNA-184 regulates angiogenic factors to maintain corneal avascularity. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4495.
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microRNA-184 (miR-184), the most abundant short non-coding RNA in the corneal epithelium, antagonizes miR-205 to maintain SHIP2 levels. Preserving SHIP2 levels, which down-regulates the Akt pathway, may reflect the need for corneal avascularity. To further evaluate the contribution of miR-184 to corneal avascularity, we investigated the role of miR-184 as an angiostatic factor.
Total RNAs were isolated from human limbal and corneal epithelia following laser capture microdissection. Expression levels of miR-184 in these tissues as well as in primary cultured limbal (HLEKs) and corneal (HCEKs) epithelial keratinocytes were measured by TaqMan qPCR assay. miR-184 expression was also determined by in situ hybridization in human and mouse cornea/limbus. mRNA expression was evaluated by qRT-PCR. The effects of miR-184 on scratch wound healing of human dermal microvascular endothelial cells (HDMECs) were measured using conditioned media. In silico, cell-based luciferase reporter assays and immunoblotting were used to validate potential targets of miR-184.
miR-184 expression in corneal epithelium and HCEKs was 10- and 4-fold higher than in limbal epithelium and HLEKs, respectively. In situ hybridization confirmed that miR-184 was strongly expressed in the corneal epithelium compared to limbal epithelium. Scratch wounding of HDMECs demonstrated a 35% reduction in closure in media conditioned by HLEKs expressing miR-184 compared with control conditioned media. This indicates that miR-184-expressing cells secrete lower amounts of trophic factors including angiogenic mitogens than control cells. Phospho-Akt, which is involved in VEGF secretion and angiogenesis, was markedly decreased in HCEKs expressing miR-184 compared with controls. Soluble Flt-1 was reduced in HCEKs treated with antago-184 compared with an irrelevant control. Luciferase assay and protein data indicated that miR-184 targets multiple pro-angiogenic molecules, including splicing factor-1 (sf-1), platelet-derived growth factor beta (PDGF beta), friend of gata-2 (fog-2) and nuclear undecaprenyl pyrophosphate synthase 1 (nus1).
miR-184 functions to inhibit angiogenesis via negative control of multiple pro-angiogenic factors and maintains angiostasis/avascularity. The use of miR-184 to modulate angiogenesis may be a novel therapeutic strategy to prevent neovasculaturization in the cornea.
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