The Protective Role Of MiR-34c-regulated Autophagy In Diabetic Keratopathy

Purpose Corneal nerve damage is one of high risk factors of corneal disease.Full elucidation of the pathogenesis of corneal nerve damage provides a new persepective for the comprehensive elucidation of the pathogenesis of diabetic neuropathy.In this study,we researched micro-RNA(mi RNA)directly targeting Atg4 B gene to achieve the protective effect and molecular mechanism of diabetic peripheral neuropathy.Methods In this study,we used streptozotocin(STZ)-induced type I diabetes C57 mice as animal models,and first we detected the expression of mi R-34 c and autophagic intensity in trigeminal ganglion(TG)tissue.Use bioinformatics software to predict and analyzes the potentcial targets of mi R-34 c.Atg4B was selected as the target gene of mi R-34 c.It was verified by luciferase report gene detection technology and re-verified on TG nerve cells cultured in vitro.Primary trigeminal ganglion neurons were cultured in vitro to investigate the effect of mi R-34 c on the axon growth and survival of TG cells.The corneal epithelial damage-healing model of normal and diabetic mice were established and administrated with subconjunctival injection at once which divided into normal group?diabetic group?diabetic with mi R-34 c antagomir gropu and diabetic with mi R-34 c antagomir NTC.The condition of corneal epithelial healing was observed by sodium fluorescein staining,and the peripheral nerve degeneration of the cornea was evaluated by ?-tublin corneal nerve staining.The TG tissues were harvested after 72 hours to detected of the expression of autophagy-related protein by Western-blot and immunofluorescence co-localization technique.Results Compared with normal C57 mice,streptozotocin(STZ)-induced type I diabetes C57 mice showed basic characteristics such as polydipsia,polyphagia,polyuria and persistent hyperglycemia.The expression of mi R-34 c was significantly increased in TG tissue of type I diabetic mice by the real-time PCR.Western-blot and immunofluorescence co-localization technique showed that autophagy-related proteins Atg4 B and LC3-II were significantly down-regulated in diabetes TG compared with normal control.Atg4 B was predicted to be the most putative target gene of mi R-34 c by Targetscan software.Luciferase report assay and western-blot comfired mi R-34 c could silenced expression of Atg4 B.Treatment of primary cultured trigeminal ganglion cells with mi R-34 c antagomir showed that the length of the trigeminal ganglion cell synapses was significantly increased after mi R-34 c antagomir treatment compared with normal cultured trigeminal ganglion cells.Subconjunctival injection of mi R-34 c antagomir can significantly promoted the corneal epithelium healing of diabetic mice,and significantly promoted the regeneration of corneal nerve.At the same time,it can significantly increased the expression of autophagy in TG tissue of type I diabetic mice.Conclusion The expression of mi R-34 c in trigeminal ganglion tissue of type I diabetic mice was up-regulated and autophagy was weakened.Inhibition of mi R-34 c expression can affect the growth of trigeminal sensory neurons and the repair of diabetic corneal nerve endings by acting directly on Atg4 B.This study suggests that mi R-34 c plays an important role in the pathogenesis of diabetic diabetic peripheral neuropathy.