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Jong Kook, Han Peng, Wending Yang, Olga Volpert, Robert M Lavker; miR-184 directly regulates corneal angiogenesis. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3513. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Due to the need for avascularity, much effort has been made to unravel the biology of corneal angiogenesis and angiostasis. However, how corneal avascularity is regulated and maintained is still incomplete. We proposed that microRNA-184 (miR-184), the most abundant, short non-coding RNA in corneal epithelium, indirectly preserved corneal avascularity due to ability to negatively regulate Akt signaling, which could suppress angiogenesis. We now present direct evidence that miR-184 regulates corneal angiogenesis.
Matrigel plugs containing media conditioned by primary cultured human limbal epithelial keratinocytes (HLEKs) engineered to over-express miR-184 were compared with control conditioned media. Angiogenesis was also evaluated in Matrigel plugs containing media conditioned by primary cultured human corneal epithelial keratinocytes (HCEKs) treated with antagomir-184 (antago-184) to knock down miR-184. Samples were recovered 5 days after subcutaneous implantation into mice and microvessel density was measured using a mouse endothelial cell specific anti-CD31 antibody. miR-184 targets were analyzed by immunofluorescence staining of sections of human anterior segmental epithelium. Loss-of-function experiments in HLEKs were complemented by siRNA against the miR-184 targets to assess biological function.
A significantly fewer number of blood vessels per area were formed in Matrigel plugs containing media conditioned by HLEKs overexpressing miR-184 (15 ± 0.6) compared with control conditioned media (23 ± 3). Conversely, a marked two fold increase in neovascularization was detected in plugs harboring conditioned media from antago-184-treated HCEKs (27 ± 2.5 vs 14.3 ± 4.2). Platelet-derived growth factor β (PDGF β), friend of gata-2 (fog-2) and nuclear undecaprenyl pyrophosphate synthase 1 (nus1), all direct targets of miR-184, were minimally detected in corneal epithelium, but highly expressed in limbal epithelium. Knockdown of fog-2 in HLEKs resulted in a down-regulation of VEGF-A mRNA levels. Silencing nus1 genetically in HLEKs decreased the expression of the pro-angiogenic factors, p-Akt and fatty acid synthase.
Our findings provide strong evidence that miR-184 functions to inhibit corneal vascularization via negative regulation of PDGF β, fog-2 and nus1, well known pro-angiogenic factors. Such negative regulation implicates miR-184 as a major contributor to the angiostatic/avascularity nature of the cornea.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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