July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Proteomics profiling to elucidate miR-146a targets in primary limbal epithelial cells
Author Affiliations & Notes
  • Mehrnoosh Saghizadeh
    Biomedical Sciences, Regenerative Medicine Institute Eye program, Cedars-Sinai Medical Center, Los Angeles, California, United States
    David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States
  • Adam Poe
    Biomedical Sciences, Regenerative Medicine Institute Eye program, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Mangesh Kulkarni
    Biomedical Sciences, Regenerative Medicine Institute Eye program, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Andrei A Kramerov
    Biomedical Sciences, Regenerative Medicine Institute Eye program, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Alexander V Ljubimov
    Biomedical Sciences, Regenerative Medicine Institute Eye program, Cedars-Sinai Medical Center, Los Angeles, California, United States
    David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States
  • Yasaman Jami-Alahmadi
    Department of Biological Chemistry, University of California Los Angeles, Los Angeles, California, United States
    David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States
  • James Wohlschlegel
    Department of Biological Chemistry, University of California Los Angeles, Los Angeles, California, United States
    David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Mehrnoosh Saghizadeh, None; Adam Poe, None; Mangesh Kulkarni, None; Andrei Kramerov, None; Alexander Ljubimov, None; Yasaman Jami-Alahmadi, None; James Wohlschlegel, None
  • Footnotes
    Support  NIH R01 EY025377
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 913. doi:
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    • Get Citation

      Mehrnoosh Saghizadeh, Adam Poe, Mangesh Kulkarni, Andrei A Kramerov, Alexander V Ljubimov, Yasaman Jami-Alahmadi, James Wohlschlegel; Proteomics profiling to elucidate miR-146a targets in primary limbal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):913.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We have previously shown the regulatory role of miR-146a in human corneal epithelial wound healing and its upregulation in limbus vs.central cornea and also in diabetic vs.normal limbus. The purpose was to identify direct and/or downstream targets of miR-146a using protein quantification in human primary limbal epithelial cells (LEC).

Methods : Primary LEC were transfected with select miR-146a mimic (M) and its respective control (MC). Three days after transfection, the cells were collected, and cell lysates prepared for proteomics analysis. We used liquid chromatography/mass spectrometry global proteomic approach,tandem mass tagging (TMT) method.Labeled samples using 10-plex TMT isobaric tags were mixed and fractionated offline using high pH reversed phase chromatography. Individual fractions were analyzed by LC-MS/MS using online reversed phase chromatography and tandem mass spectrometry on a Thermo Fisher Fusion Lumos mass spectrometer. Data were acquired using the synchronous precursor selection-based MS3 method. Database searching, and the extraction of TMT reporter ion information was performed using the Proteome Discoverer software. Comparison of TMT data across samples was performed using MSStats.In addition, expression data were analyzed in conjunction with TargetScan to increase the likelihood of finding direct miRNA targets.

Results : We identified 101 proteins with expression changes of >2.0-fold and p value < 0.05 in the miR-146a mimic vs.mimic control transfected primary LEC. Overexpression of miR-146a in LEC significantly decreased protein levels of some known or predicted miR-146a targets EGFR, TRAF6 and NUMB and proinflammatory cytokines such as IL-1β and IL-1α compared to control. Interestingly, NF-kB inhibitor and putative limbal epithelial stem cell marker, keratin 15, showed upregulation upon miR-146a overexpression. The most significantly changed pathways for the target proteins found to be differentially expressed in M vs.MC included p38 MAPK, extracellular matrix organization, integrin and VEGF signaling pathways.

Conclusions : Proteomics approach may help unveil the molecular targets and potential mechanisms and pathways underlying miR-146a functions in human limbal epithelial cells.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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