Integrated genomics and proteomics analysis reveal the regulatory role of miR-10b targets in normal and diabetic human limbal epithelial cells

Abstract

Purpose : MicroRNA-10b (miR-10b) is up-regulated in the limbus vs central human cornea and also in diabetic (DM) vs normal (N) limbus, which may contribute to limbal cell function and diabetic corneal disease, respectively. Our aim was to identify miR-10b target genes/proteins using integrated genomic and proteomic analyses to elucidate its role in the normal and diabetic limbus

Methods : Human autopsy N and DM corneas were procured by the National Disease Research Interchange (NDRI) in Optisol medium. In vitro experiments were performed with stem cell-enriched human N and DM primary limbal epithelial cells (LECs). miRNA transfection was performed using Lipofectamine RNAiMAX. Normal LECs were transfected with miR-10b mimic (M), or its mimic control (MC). At day 3 post-transfection, cells were harvested for total RNA isolation using Trizol for next-generation RNA sequencing (RNA-seq). A matching set of transfected LECs was collected and cell lysates were prepared for proteomics analysis

Results : Sets of 661 mRNAs (FC of ± 2, 305 up- and 356 down-regulated) and 144 proteins (FC of ± 1.5, 72 up- and 72 down-regulated) were identified as differentially expressed in miR-10bM vs. MC with adjusted p < 0.05 using RNA-seq and proteomics analyses, respectively. Combined proteomics and genomics analysis revealed a total of 17 overlapping genes as potential targets of miR-10b, all considered putative or validated targets in the literature. Additionally, pathway analysis of proteomics and transcriptomics data revealed significant changes in multiple signaling pathways such as cell cycle, DNA repair and replication, Wnt and TP53 signaling. Overexpression of miR-10b in primary N and DM LECs significantly decreased expression levels of target proteins GCLM, CMPK1, Lig1, and TPM4 by western blot and immunostaining. In addition down-regulation of miR-10b in DM organ-cultured corneas using miR-10b inhibitor increased expression levels of TPM4 and Lig1 involved in DNA repair and replication by immunostaining

Conclusions : In conclusion, by using integrated genomic and proteomic analyses of cultured N and DM LECs, potential key target genes of miR-10b were revealed as well as the underlying pathways affecting human LEC function. Further analysis of promising targets may improve our understanding of the molecular mechanisms underlying diabetic corneal alterations and wound healing

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.