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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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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).
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.
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.
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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