Oxidative stress-regulatory role of miR-10b-5p in the diabetic human cornea revealed through integrated multi-omics analysis.
Publication Year:
2026
PubMed ID:
41115935
Funding Grants:
Public Summary:
Diabetes can affect the surface of the eye (the cornea) in several ways. It can increase harmful stress in cells, damage nerves, and make it harder for the eye to heal properly. It may also interfere with special stem cells at the edge of the cornea that are important for maintaining healthy tissue.
In this study, we focused on tiny molecules called microRNAs—specifically one called miR-10b-5p—that may play a role in these problems. We wanted to understand how higher levels of this molecule in people with diabetes affect corneal cells compared to those without diabetes. To do this, we looked at which genes and proteins it influences and explored whether targeting it could help reduce cell stress and improve eye health in diabetic patients.
Scientific Abstract:
AIMS/HYPOTHESIS: Prominent features of diabetic corneal disease are oxidative stress, neuropathy and epitheliopathy including delayed wound healing and dysfunction of limbal epithelial stem cells. We hypothesised that regulatory miRNAs altered in the diabetic cornea, such as miR-10b-5p, may be responsible for these abnormalities. We aimed to understand the molecular impact of miR-10b-5p increase in human diabetic vs non-diabetic limbal epithelial cells (LECs) enriched in limbal epithelial stem cells by identifying its target genes and proteins and testing it as a potential therapy for inhibiting oxidative stress in diabetic corneas. METHODS: LECs were isolated from diabetic and non-diabetic human autopsy corneas. Telomerase-immortalised human corneal epithelial cells (HCECs), primary LECs and ex vivo organ-cultured corneas were transfected with 50 nmol/l hsa-miR-10b-5p mimic or miRNA inhibitor or siRNA against GCLM (glutamate-cysteine ligase modifier subunit) along with their respective controls using Lipofectamine RNAiMAX. Total RNA was extracted for transcriptomic analysis. Proteins were extracted, digested and quantified using LC-MS/MS proteomics. Oxidative stress was induced using 200 micromol/l hydrogen peroxide (H(2)O(2)) in transfected LECs and/or HCECs post starvation. Cell lysates at 0, 3, 6, 9 and 24 h time points were analysed on western blots. Reactive oxygen species in transfected HCECs were measured using the DCFDA/H2DCFDA-Cellular ROS Assay Kit. Glutathione (GSH) levels were quantified using the GSH-Glo assay from H(2)O(2)-treated LECs. Glutamate-cysteine ligase modifier subunit (GCLM) and lanthionine synthetase C-like protein 1 (LANCL1) protein expression levels were also analysed by immunostaining. RESULTS: Integrative proteomic and genomic analysis of miR-10b- vs miRNA mimic control-transfected primary LECs identified GCLM and LANCL1 as key miR-10b-5p targets (false discovery rate p<0.05), validated by western blot and immunostaining. miR-10b- and siRNA-GCLM-transfected LECs 3 h after H(2)O(2) treatment showed a significant reduction in glutathione/glutathione disulfide (2GSH/GSSG) ratio and overall GSH levels. Further, miR-10b-5p-transfected HCECs produced higher ROS levels, peaking at 12.67 +/- 0.22% at 6 h post H(2)O(2) treatment, as compared with 10.41 +/- 0.20% in controls. This implicates downregulated LANCL1 in modulating cellular responses to oxidative damage. Both GCLM and LANCL1 were downregulated in ex vivo diabetic corneas, while inhibition of miR-10b-5p significantly restored their expression in diabetic organ-cultured corneas by immunostaining. CONCLUSIONS/INTERPRETATION: Our findings suggest that diabetes-overexpressed miR-10b disrupts redox balance by targeting GCLM and LANCL1, which potentially leads to increased oxidative stress and cellular vulnerability in diabetic corneas. Inhibiting miR-10b-5p restored antioxidant defences, suggesting a potential therapeutic strategy to mitigate oxidative stress and normalise corneal health in individuals with diabetes and preserve vision.