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Mehrnoosh Saghizadeh, Mangesh Kulkarni, Aleksandra Leszczynska, Jie Tang, Talia Barkhordari, Nima Natanzi, Alexander V Ljubimov, Vincent A Funari; Essential Role of miR-146a in Limbal Epithelial Stem Cell Maintenance via Notch Signaling. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2991.
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We have previously shown upregulation of miR-146a in limbus vs. central cornea and also in diabetic vs. normal limbus, and its role in wound healing. The study purpose was to evaluate the effects of miR-146a on corneal limbus, especially in relation to limbal epithelial stem cells (LESC).
Human autopsy normal corneas were received from the National Disease Research Interchange (NDRI) in Optisol storage medium. The in vitro experiments were performed with either Stem cell-enriched primary limbal epithelial cells (LEC) or telomerase-immortalized human corneal epithelial cells (HCEC). Transfection was performed using Lipofectamine RNAiMAX (Thermo Fisher) following the manufacturer’s instructions. Next generation mRNA sequencing was performed on total RNA extracted from primary LEC treated with miR-146a mimic (for overexpression), inhibitor (for inhibition) and their corresponding controls. Western blot was performed to confirm the effects of miR-146a at the protein level.
mRNA sequencing showed that miR-146a had a profound effect on Notch signaling in corneal limbus. miR-146a overexpression led to a decrease in mRNA levels of Numb and Notch-2 whereas Notch-1 mRNA level was increased. We further investigated these effects at the protein level in HCEC and in ex vivo corneal organ cultures. We found that Numb, a Notch inhibitor, is a direct target of miR-146a both in vitro and in ex vivo organ-cultured corneas. miR-146a-induced repression of Numb led to an increase in Notch-1 expression level. Interestingly, in agreement with sequencing data, Notch-2 protein level was decreased by miR-146a overexpression. In addition, miR-146a overexpression led to an increased expression of limbal epithelial stem cell marker, keratin 15 (K15).
In the cornea, miR-146a plays critical role in stem cell maintenance by regulating Notch signaling. This is supported by increased expression of K15 by miR-146a overexpression. Studies are underway to investigate miR-146a and downstream effects of Notch signaling in diseased corneas, which are associated with stem cell dysfunction.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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