| Objective:Diabetic keratopathy(DK)is one of the main eye complications in diabetic patients.Due to the increasing incidence of diabetes,DK has received increasing attention.To study the role of micro RNA 21(miR-21)in diabetic keratopathy and explore its mechanism is expected to provide new ideas for the treatment of DK.Methods:The expression of miR-21 in corneal epithelium and trigeminal ganglia tissues of normal mice and diabetic mice was compared by RT-PCR.Corneal epithelial injury models of WT and miR-21 KO diabetic mice were established.The healing of corneal epithelium was observed by fluorescin sodium staining,and regeneration of corneal subepithelial nerve plexus was evaluated by specific nerve staining.TUNEL fluorescence staining was used to detect the apoptosis of corneal epithelium and trigeminal ganglia tissues.Mi R-21 was overexpressed in mouse corneal stem/progenitor cell lines(TKE2)and treated with high glucose.Cell migration was detected by scratch assay,and cell apoptosis was detected by Annexin V/7-AAD double fluorescence staining.Mi R-21 was overexpressed in primary trigeminal ganglion cells,and the axonal growth of trigeminal ganglion cells was determined by immunofluorescence staining,and the apoptosis of trigeminal nerve cells was detected by TUNEL staining.Genes differentially expressed in corneal epithelium and trigeminal ganglia tissues of WT and miR-21 KO diabetic mice were screened by transcriptome sequencing,and biofunction enrichment analysis of differentially expressed genes was performed by GO analysis.The corneal epithelial injury model was established in diabetic mice subconjunctivally injected with miR-21 agomir or control agomir.The recovery of corneal epithelial injury and the regeneration of corneal peripheral nerves were determined.The apoptosis of corneal epithelium and trigeminal ganglia tissue was detected by TUNEL staining.Results:The expression of miR-21 was increased in corneal epithelium and trigeminal nerve tissue of diabetic mice.Mi R-21 was also significantly up-regulated in the regenerated epithelium after corneal epithelial injury,but its increase in the corneal epithelium of diabetic mice was not as obvious as that of control mice;The recovery rate of corneal epithelial injury and the density of corneal nerve regeneration in miR-21 KO diabetic mice were significantly slower than that in WT diabetic mice.Further studies showed that the apoptosis of corneal epithelial cells and trigeminal nerve was increased in miR-21 KO diabetic mice compared with that in WT diabetic mice.After the overexpression of miR-21,the migration ability of TKE2 cells under high glucose condition and the axon length of primary trigeminal nerve cells were significantly increased compared with the control group,and the apoptosis of TKE2 cells and trigeminal nerve cells was decreased.Through transcriptome sequencing analysis,497 and 554 genes were found differentially expressed in corneal epithelial tissue and trigeminal nerve tissue between WT and miR-21 KO diabetic mice,respectively.GO analysis showed that the signal pathways regulated by differentially expressed genes were mainly enriched in cell migration,cell adhesion,cell proliferation and differentiation,cell apoptosis,injury repair and nerve growth.The subconjunctival injection of miR-21 agomir in diabetic mice significantly accelerated the recovery of corneal epithelial injury and promoted corneal nerve regeneration.Conclusion:1.Corneal epithelial injury leads to increased expression of miR-21 in corneal epithelial tissues,but the increase of miR-21 in corneal epithelial tissues of diabetic mice was less than that of normal mice;2.Mi R-21 promotes the migration of corneal epithelial cells,inhibits the apoptosis of corneal epithelial cells and trigeminal nerve cells induced by high glucose,and promotes nerve growth;3.Overexpression of miR-21 in diabetic mice can promote the recovery of corneal epithelial injury and regeneration of corneal peripheral nerves. |
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