June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Parallel genomic and proteomic profiling of miR-10b targets in human limbal epithelial cells
Author Affiliations & Notes
  • Adam Poe
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Regenerative Medicine Institute Eye Program, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • DRIRH KHARE
    Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Regenerative Medicine Institute Eye Program, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Yizhou Wang
    Genomics Core, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Alexander V Ljubimov
    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
  • Jennifer Van Eyk
    Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States
    Advanced Clinical Biosystems Research Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States
  • Mehrnoosh Saghizadeh
    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
  • Footnotes
    Commercial Relationships   Adam Poe, None; DRIRH KHARE, None; Yizhou Wang, None; Alexander Ljubimov, None; Jennifer Van Eyk, None; Mehrnoosh Saghizadeh, None
  • Footnotes
    Support  NIH R01 EY025377, R01 EY029829 (Saghizadeh), and Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center.
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 868. doi:
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      Adam Poe, DRIRH KHARE, Yizhou Wang, Alexander V Ljubimov, Jennifer Van Eyk, Mehrnoosh Saghizadeh; Parallel genomic and proteomic profiling of miR-10b targets in human limbal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2021;62(8):868.

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

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Abstract

Purpose : We previously showed that microRNA-10b (miR-10b) is one of the most abundant limbal miRNAs, which is upregulated in the limbus vs. central cornea and may regulate corneal epithelial homeostasis and stem cell functions. Additionally, we showed its upregulation in diabetic (DM) vs. normal limbus, which may contribute to diabetic corneal disease. Our purpose was to investigate miR-10b target genes and regulated proteins using combined genomic and proteomic analyses to elucidate its role in the limbus.

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 human primary limbal epithelial cells (LECs). LECs were transfected with either miR-10b mimic, inhibitor, or their corresponding controls using Lipofectamine RNAiMAX (Thermo Fisher) following the manufacturer’s instructions. At day 3 post-transfection cells were harvested for total RNA isolation using Trizol for next generation mRNA sequencing. A matching set of transfected cells was collected and analyzed by global proteomic analysis.

Results : Proteomics and mRNA-seq elucidated changes in pathways regulating limbal epithelial stem cells (LESC) maintenance and differentiation as a result of miR-10b overexpression in LECs. Our previous findings indicated major transcription factors controlling eye development and maintenance of the cornea as potential targets of miR-10b. Our current study found that miR-10b mimic downregulates KLF4, RAP2A, and EPHB2 that regulate epithelial-mesenchymal transition. Pathway analysis indicated a role in cell cycle regulation because of changes in related predicted miR-10b targets — E2F7 and CDK6. Previous findings showed that miR-10b increased LEC proliferation. Transfection with miR-10b resulted in downregulation of DKK1, GSK3B, CEMIP, and NEDD4/L that regulate canonical Wnt signaling pathway. There was a good concordance between the two methods.

Conclusions : Our data revealed potential direct targets of miR-10b and unveiled the underlying pathways affecting human limbal epithelial cell maintenance, proliferation and self-renewal. Combined approach of proteomics and genomics profiling provides insight into the regulation by microRNAs of their targets on a genome/proteome-wide scale.

This is a 2021 ARVO Annual Meeting abstract.

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