The Function And Mechanism Of Mir-18a In Promoting Corneal Epithelial Wound Healing By Sodium Hyaluronate

IntroductionProperly controlled corneal epithelial wound healing is critical for health of cornea,which involves cell proliferation,migration,anchoring and differentiation.Sodium hyaluronate(SH)has been proven to exert beneficial pharmacological effect on corneal wound healing,though the underlying mechanism remained open to investigation.In this light,we intend to detect in vitro whether miRNAs are also implicated in the mechanism underlying the stimulatory effect of SH on corneal epithelial wound healing.Chapter1 Expression profile of miRNA in sodium haluronate treated human corneal epithelial cellsObjectiveTo investigate the expression profile of miRNA in sodium haluronate treated human corneal epithelial cells(HCECs).MethodsHCECs were assigned to control or experimental group.Control group and experimental group were cultured in KGM-2 alone and KGM-2 with SH(0.6mg/ml),respectively.Finally 100ng of total RNA was processed for use on the microarray.Differentially expressed microRNAs were defined as log2fold chane?1.5 or?-1.5(adjP<0.05).ResultsThere was only a few significant variation in treated cells compared with control.In total,16 miRNAs were deregulated,among which miR-18a and miR-92 were most highly downregulated,and miR-210,miR-26b,and miR-378 showed the largest fold change.RT-qPCR was performed to conform the miR-18a expression,which was dramatically repressed(logFC(log-transformed fold change)=-1.987983,p=2.05E-13),next to which was miR-92(logFC=-1.228615,p=6.52E-12).ConclusionsmiR-18a are significantly downregulated in sodium hyaluronate treated human corneal epithelial cells.Chapter 2 Effects of miR-18a on biological characteristic of human corneal epithelial cellsObjectiveTo investigate the effects of miR-18a on biological characteristic of HCECs.MethodsScrambled control or miR-18a antagomir/mimic were introduced into HCECs using Lipofectamine RNAi Max.Cells were harvested for RT-qPCR and for MTT assay 24h,48h,72h after transfection.The absorbance at 570nm were taken during the cell viability assay.ResultsThe transfection efficiency was confirmed by quantifying miR-18a for every treatment or transfection.The HCECs transfected with miR-18a mimics showed cell growth rate slower than HCECs transfected with scramble sequences,while those transfected with miR-18a antagomir displayed accelerated growth rate.ConclusionsmiR-18a is identified as effector in the stimulatory effect of SH on HCECs proliferation.Chapter 3 miR-18a promotes proliferation and migration of sodiumhyaluronate treated human corneal epithelial cells by targeting CTGFObjectiveTo predict the probable target gene of miR-18a and to conform it.To investigate regulating effect of target gene.MethodsTargetScan and functional enrichment analysis were employed to predict targets of miR-18a.Scrambled control,SH+Scrambled siRNA,miR-18a antagomir/mimic were introduced into HCECs using Lipofectamine RNAi Max.RT-qPCR and Western blot were performed to detect the expression of target gene.Dual-luciferase activity assay were conducted to verify whether the target gene is a bona fide target of miR-18a.Transfected the siRNA of the target gene in the SH treated HCECs.RT-qPCR and Western blot were performed to comfirm the knockdown efficiency.MTT assay,transwell migration and scratch wound healing were used to investigate the role of the target gene in SH treated HCECs.ResultsTargetScan and functional enrichment analysis showed that the putative targets of miR-18a were enriched in Arf6 downstream pathway.miR-18a was postulated to modulate biological processes by suppressing CTGF.The transfection efficiency was confirmed by quantifying miR-18a for every treatment or transfection.Transfection with miR-18a antagomir resulted in a remarkable increase in CTGF protein,while the HCECs transfected with miR-18a mimics present substantial reduction.In addition,the level of CTGF in SH treated cells was dramatically elevated compared to untreated cells(scrambled control).HCEC cells transfected with miR-18a mimics and WT-CTGF 3'UTR had luciferase activity significantly lower than those transfected with miR-18a mimics and MT-CTGF 3'UTR.CTGF silenced cells displayed slower growth than control.In transwell migration and wound healing,CTGF ablated HCECs showed impaired cell mobility.ConclusionsCTGF is a target of miR-18a.Silencing of CTGF counteracted the migration and proliferation enhanced by SH.Downregulation of miR-18a induces CTGF and promotes proliferation and migration of SH treated HCECs.