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Adam Poe, Mangesh Kulkarni, Jason Wang, Aleksandra Leszczynska, Jie Tang, Alexander V Ljubimov, Mehrnoosh Saghizadeh; miR-146a regulatory roles in limbal epithelial homeostasis. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4826.
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© ARVO (1962-2015); The Authors (2016-present)
We previously showed that miR-146a upregulation significantly altered corneal epithelial wound healing and expression of putative limbal epithelial cell (LEC) markers. The purpose was to investigate the regulatory roles of miR-146a targets in normal corneal limbus. We evaluated the effects of miR-146a on Notch signaling and inflammatory pathways with or without lipopolysaccharide (LPS) treatment.
Human autopsy normal corneas were procured from the National Disease Research Interchange in Optisol medium. In vitro experiments were performed with stem cell-enriched LECs. MicroRNA transfection was performed using Lipofectamine RNAiMAX (Thermo Fisher) following the manufacturer’s instructions. Next generation RNAseq was performed on total RNA from primary LEC treated with miR-146a mimic, inhibitor and their corresponding controls. Acute inflammatory response was induced by 10 ng/ml LPS for 24h. Western blot analysis was used to confirm changes in miR-146a target proteins.
Next generation RNAseq revealed several genes as possible targets of miR-146a. Overexpression of miR-146a decreased mRNA levels of Numb and Notch-2, while Notch-1 levels increased. In line with sequencing data, Numb and Notch-2 protein levels were decreased by miR-146a overexpression. In addition, miR-146a overexpression led to an increased expression of LESC marker, keratin 15 (K15). Overexpression of miR-146a also markedly decreased mRNA levels of NF-kB upstream regulators, TRAF-6 and IRAK-1. To explore this effect, we induced inflammation by LPS in LEC in vitro and in ex vivo organ-cultured corneas, which led to increased expression of TRAF-6 and IRAK-1 proteins. MiR-146a overexpression suppressed TRAF-6 and IRAK-1, whereas its inhibition led to the increase of TRAF-6 and IRAK-1 in LPS treated LEC.
miR-146a plays an important role in limbal epithelial cell maintenance via regulation of the Notch signaling pathway, supported by upregulation of K15 by miR-146a overexpression. Further, miR-146a regulates the NF-kB inflammatory response by targeting Traf6 and Irak1. Under inflammatory conditions, miR-146a may suppress inflammatory mediators through the NF-kB pathway. Additionally, its downregulation of Numb, which is an inflammation inducer, may also contribute to its anti-inflammatory effects.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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