Parallel genomic and proteomic profiling of miR-10b targets in human limbal epithelial cells

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.