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Integrated genomics and proteomics analysis uncovers the oxidative stress role of miR-10b targets in healthy and diabetic human limbal epithelial cells

ABSTRACT: The corneal epithelium is maintained by limbal epithelial stem cells (LESCs) and is largely responsible for corneal optical transparency and protection by continuously renewing population of corneal epithelial cells. Diabetes mellitus (DM) affects all structures of the eye including the cornea, which can result in delayed wound healing and potential vision loss. MicroRNAs (miRNAs) are short non-coding oligonucleotides that regulate various cellular functions, including oxidative stress response, by repressing protein translation. MiR-10b-5p was previously identified to be upregulated in diabetic vs. non-diabetic limbal cells, and our purpose was to understand the role of miR-10b-5p in human limbal epithelial cells in healthy and diabetic conditions. Through integrated transcriptomic and proteomic analyses, we identified GCLM and LANCL1 as key miR-10b-5p targets, revealing its profound impact on glutathione metabolism, sulfur compound biosynthesis processes, and antioxidant defenses. Our findings suggest that overexpression of miR-10b disrupts redox balance, which potentially leads to heightened oxidative stress and increased cellular vulnerability in diabetic corneas. Understanding miR-10b function in corneal epithelial cells may pave the way for novel therapeutic strategies to mitigate oxidative stress and normalize corneal health in diabetic patients.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Mehrnoosh Saghizadeh Ghiam

PROVIDER: MSV000097328 | MassIVE | Fri Mar 14 10:58:00 GMT 2025

SECONDARY ACCESSION(S): PXD061853

REPOSITORIES: MassIVE

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Integrated genomics and proteomics analysis uncovers the oxidative stress role of miR-10b targets in healthy and diabetic human limbal epithelial cells

Project description:The corneal epithelium is maintained by limbal epithelial stem cells (LESCs) and are largely responsible for corneal optical transparency and protection by continuous population of corneal epithelial cells. Diabetes mellitus (DM) has been shown to affect all structures of the eye including the cornea which can result in delayed wound healing and potential vision loss. MicroRNAs (miRNAs) are short non-coding oligonucleotides that regulate various cellular functions, including oxidative stress response, by repressing translation. miR-10b-5p was previously identified to be upregulated in diabetic vs. non-diabetic limbal cells and our purpose was to understand the role of miR-10b-5p in human limbal epithelial cells in normal and pathological conditions such as diabetes mellitus. It was hypothesized that miR-10b-5p influences corneal epithelial homeostasis by modulating antioxidant defenses and wound healing processes. Through integrated transcriptomic and proteomic analyses, we identified GCLM and LANCL1 as key miR-10b-5p targets, revealing its profound impact on glutathione metabolism, sulfur compound biosynthesis processes, and antioxidant defenses. Our finding suggests that miR-10b suppression disrupts redox balance which potentially leads to heightened oxidative stress, impaired wound healing and increased cellular vulnerability in diabetic corneas. Further research into miR-10b inhibitors or gene modulation strategies may pave the way for novel treatments aimed at mitigating oxidative stress-related corneal dysfunction, particularly in diabetes-induced ocular diseases.

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Project description:MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples.

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