Integrated Transcriptome and Proteome Analyses Reveal the Regulatory Role of miR-146a in Human Limbal Epithelium via Notch Signaling.

Adam J Poe,Ruchi Shah,James Wohlschlegel,Yasaman Jami-Alahmadi,Andrei A Kramerov,Mehrnoosh Saghizadeh,Alexander V Ljubimov,Vasu Punj,Mangesh Kulkarni,Jason Wang,Jie Tang,Aleksandra Leszczynska

doi:10.3390/cells9102175

Abstract

MiR-146a is upregulated in the stem cell-enriched limbal region vs. central human cornea and can mediate corneal epithelial wound healing. The aim of this study was to identify miR-146a targets in human primary limbal epithelial cells (LECs) using genomic and proteomic analyses. RNA-seq combined with quantitative proteomics based on multiplexed isobaric tandem mass tag labeling was performed in LECs transfected with miR-146a mimic vs. mimic control. Western blot and immunostaining were used to confirm the expression of some targeted genes/proteins. A total of 251 differentially expressed mRNAs and 163 proteins were identified. We found that miR-146a regulates the expression of multiple genes in different pathways, such as the Notch system. In LECs and organ-cultured corneas, miR-146a increased Notch-1 expression possibly by downregulating its inhibitor Numb, but decreased Notch-2. Integrated transcriptome and proteome analyses revealed the regulatory role of miR-146a in several other processes, including anchoring junctions, TNF-α, Hedgehog signaling, adherens junctions, TGF-β, mTORC2, and epidermal growth factor receptor (EGFR) signaling, which mediate wound healing, inflammation, and stem cell maintenance and differentiation. Our results provide insights into the regulatory network of miR-146a and its role in fine-tuning of Notch-1 and Notch-2 expressions in limbal epithelium, which could be a balancing factor in stem cell maintenance and differentiation.

Highlights

The corneal epithelium is continuously renewed by expansion of limbal epithelial stem cells (LESCs) residing at the corneoscleral junction, the limbus [1,2]
To better understand the regulatory role of miR-146a in the limbal epithelium, we have investigated the potential targets of miR-146a in limbal epithelial cells (LECs) using RNA-seq transcriptomics combined with quantitative proteomics analysis based on multiplexed isobaric tandem mass tag (TMT) labeling
To identify miR-146a target genes, primary human LECs from individual donors (n = 8) were transfected with miR-146a mimic and its corresponding negative control

Summary

Introduction

The corneal epithelium is continuously renewed by expansion of limbal epithelial stem cells (LESCs) residing at the corneoscleral junction, the limbus [1,2]. As part of regular corneal epithelial homeostasis, LESCs give rise to transient amplifying (TA) cells, which migrate towards the central cornea. Proper renewal and healing of the corneal epithelium is required to maintain corneal transparency and protection of the eye from the external environment. Pathological conditions, including diabetes mellitus (DM), can disrupt epithelial homeostasis, resulting in abnormal epithelial self-renewal and dysfunctional wound healing. We found that the diabetic corneal epithelium has significantly decreased expression of several putative LESC markers, resulting in impaired corneal epithelial wound healing [4]. Failure of LESC maintenance and subsequent epithelial renewal can result in decrease in corneal transparency, leading to vision loss

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