| Diabetes (Diabetes Mellitus, DM) is a group of metabolic disease characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. With the incidence of diabetic patients increasing, more work are need to do on prevention and treatment of diabetes and its complications. As diabeitc corneal complications are very common in diabeitc patients. It’s always a problem that diabetic patients have suffered with the delay of corneal epithelium wound healing after eye surgery. The main clinical manifestations of diabetic keratopathy are superficial punctate keratitis or persistent epithelial defect and corneal sensation of loss. More attentions are paid to diabetic keratopathy recently. Numbers of studies have shown the abnormal thickening and functional changes in the corneal epithelial basement membrane. As the pathogenesis of diabetic keratopathy is still unclear, it is a scientific problem to be solved in clinical studies.Silence signal regulatory factors1(silence signal regulating factor1, SIRTl) is a histone deacetylase involved in glucose metabolism, insulin secretion and several other metabolic pathways. SIRT1have been considered a new target for the treatment of diabetes. It has been confirmed that SIRTl expression was significantly down-regulated after overexpression of SIRT1in diabetic mice which can significantly promote corneal epithelial wound healing in diabetic corneas. However, the cause is not clear about the regulation of SIRT1.MicroRNAs (miRNAs, miRs) are a class of about22-nucleotides endogenous non-coding RNA, by binding to its target protein3’UTR region to complete the post-transcriptional level of gene regulation. MiRNAs involved in many biological processes such as development, cell death, cell proliferation and tumor formation in the central nervous system function and so on. It had numerous target genes regulated for the biological effect. There are numbers of research on miRNAs currently; however the researchs on diabetes keratopathy are not enough. Therefore, we study about the relationship between miRNAs and SIRT1on diabetic keratopathy. It may play a role in mediating expression of SIRT1in corneal epithelium.The purpose of this study is to explore the regulation between SIRTl and miRNAs which participate in diabetic keratopathy to seek effective therapeutic targets.PART1Identification of miRNA to the regulation of SIRT1in diabetic corneal epithelia by qRT-PCRPurpose:To investigate which microRNA (miRNA) in modifying the expression of Sirtuin (silent mating type information regulation2homolog)1in diabetic corneas.Methods:The bioinformatic assay was used to predict which miRNAs might regulate the expression of SIRT1. The results were verified by Quantitative Real-time PCR (qRT-PCR) in in diabetic mouse C57BL/6J-Ins2Akita(Ins2Akita/+) mice and control C57BL/5J (Ins2+/+) mice. Cell transfection protocol was used to up-regulation or knockdown the miRNA expression in mouse corneal epithelial progenitor cell line (TKE2) for the purpose of up/down regulation of miRNA.Results:Nine miRNAs were selected for qPCR detection after bioinformatics analysis. MiR-204-5p merited further investigation because it was increased5.16fold in diabetic corneal epithelia compared to non-diabetic control corneal epithelia. The mimic and inhibitor of miR-204-5p showed that it could increase/decrease the expression of SIRT1. Using luciferase activity assay, we identified SIRT1is a direct target of miR-204-5p.Conclusions:Our data provide firm evidence of a role for miR-204-5p in the direct regulation of SIRT1in diabetic corneas. The up-regulation of miR-204-5p is one of the reasons of down-regulation of SIRT1in diabetic corneas.PART2Effect of decreased miR-204-5p expression in regulation of SIRT1wih restoring cell-cycle progression in mouse corneal epithelial progenitor cell line (TKE2) in high-glucose (HG) conditionsPurpose:To observe the effect of miR-204-5p regulating SIRT1on cell-cycle status in TKE2cells in HG conditionsMethods:Adenovirus-expressing short interfering RNA was used to knockdown the expression of SIRT1in TKE2cells in HG conditions. The expression of SIRT1, Cyclin D1, p16, p21was detected by qRT-PCR. Cell-cycle status was determined by flow cytometry assay and MTT assay was used to analyze the cell viability.Results:The results of flow cytometry and MTT assay demonstrated that down-regulation of miR-204-5p increased TKE2cell growth and restores cell-cycle progression in high glucose (HG) conditions by the regulation of Cyclin D1and p16. Conclusions:MiR-204-5p mediated regulation of SIRT1contributes to the delay of epithelial cell-cycle traversal in HG condition.PART3Effect of decreased miR-204-5p expression in regulation of SIRT1with improving the wound healing in diabetic corneal epithelialPurpose:To observe the effect of miR-204-5p regulating SIRT1on wound healing in diabetic corneal epithelialMethods:We made corneal injury model in diabetic mouse C57BL/6J-Ins2Akita (Ins2Akita/+) mice and control C57BL/6J (Ins2+/+) mice. The miRNA-specific antigomir was administered by subconjunctival injection. We then observe corneal epithelial defect area in the first of0,24,48,72hours after the experiment. The expression of SIRT1, Cyclin D1, p16, p21in corneal epithelial was detected by qRT-PCR.Results:we showed down-regulation of miR-204-5p promotes high-glucose attenuation of corneal epithelial wound healing via up-regulation of SIRT1in Ins2Akita/+mice. The expression of Sirtl activated Cyclin D1/CDK1ways with up-regulaiton of Cyclin D1and down-regulaiton of p16expression decreased. Conclusions:MiR-204-5p mediated regulation of SIRT1contributes to the delay of wound healing in diabetic corneal epithelial. |
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