July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
miR-146a regulatory roles in limbal epithelial homeostasis
Author Affiliations & Notes
  • Adam Poe
    Biomedical Sciences, Cedars Sinai Medical Center, California, United States
    Regenerative Medicine Institute Eye Program, Cedars Sinai Medical Center, California, United States
  • Mangesh Kulkarni
    Biomedical Sciences, Cedars Sinai Medical Center, California, United States
    Regenerative Medicine Institute Eye Program, Cedars Sinai Medical Center, California, United States
  • Jason Wang
    Regenerative Medicine Institute Eye Program, Cedars Sinai Medical Center, California, United States
    College of Osteopathic Medicine, Touro University Nevada, Nevada, United States
  • Aleksandra Leszczynska
    Biomedical Sciences, Cedars Sinai Medical Center, California, United States
    Regenerative Medicine Institute Eye Program, Cedars Sinai Medical Center, California, United States
  • Jie Tang
    Genomics Core, Cedars Sinai Medical Center, California, United States
  • Alexander V Ljubimov
    Regenerative Medicine Institute Eye Program, Cedars Sinai Medical Center, California, United States
    David Geffen School of Medicine, University of California, Los Angeles, California, United States
  • Mehrnoosh Saghizadeh
    Regenerative Medicine Institute Eye Program, Cedars Sinai Medical Center, California, United States
    David Geffen School of Medicine, University of California, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Adam Poe, None; Mangesh Kulkarni, None; Jason Wang, None; Aleksandra Leszczynska, None; Jie Tang, None; Alexander Ljubimov, None; Mehrnoosh Saghizadeh, None
  • Footnotes
    Support  NIH R01 EY025377
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4826. doi:
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    • Get Citation

      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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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