| ObjectiveTo investigate the role of miR-665-3p in Murine Fungal Keratitis by targeting ATG5-mediated autophagy.Methods1.Target miRNA were screened and Autophagy flux was preliminarily demonstrated:First,a mouse model of Fusarium oxysporum Keratitis was established,MIR-665-3P was analyzed by gene chip and verified by QPCR,The target gene of miR-665-3p was predicted by various bioinformatics software,and ATG5 was selected as the target gene.The regulation of miR-665-3p on ATG5 was tested by Western blot and double luciferase methods in primary mouse corneal stromal cells in vitro.The morphology of autophagic bodies in infected cornea was observed by transmission electron microscope,and the expression of autophagy associated protein LC3 B and P62 were detected by Western blot.2.Cell experiment: Primary mouse corneal stromal cells were cultured in vitro and divided into 7 groups: infection group(control group),autophagy inhibition group(CQ group),autophagy activation group 1(Rapa1 group),autophagy activation Group 2(Rapa2 group),miRNA control group(miR-NTC group),miR-665-3p antagomir group(ant-665 group)and miR-665-3p agomir(miR-665 group).Treated with 50 uM chloroquine in CQ group for 12 h,treated with 100 nM rapamycin in Rapa1 group for 2h,treated with 1uM rapamycin in Rapa2 group for 2h,treated with 100 nM miR-NTC in miR-NTC group for 48 h,and treated with 100 nM miR-665-3p Antagomir treated ant-665 group for 48 h,and miR-665-3p agomir treated miR-665 group for 48 h.After each group was treated with the above-mentioned time,the cells were infected with Fusarium Solani for 6 hours at Moi=3.The expression of autophagy-related proteins LC3 B and P62 were detected by Western blot.LC3 B and lysosome were detected by co-localization of autophagy double-labeled Adenovirus.The expression of autophagic proteins ATG5 and P62 was observed by Fluorescence microscope.3.Animal experiment: Establish the mouse model of Fusarium keratitis of solanum disease,divided into 6 groups: infection group(control group),autophagy inhibition group(CQ group),autophagy activation group(Rapa group),miRNA control group(miR-NTC group),miR-665-3p antagomir group(Ant-665 group)and RapamiR-665-3p agomir(miR-665 group).After subconjunctival injection of 7ul with 50 uM CQ,1uM RAPA,100 nM miR-NTC,100 nM miR-665-3P antagomir and 100 nM miR-665-3P agomir,the mouse model of Fusarium Oxysporum Keratitis was established by modified surface microscopy.The pathological changes of cornea were observed under slit-lamp microscope 1,3,5 and 7 days after infection,and the area,degree of turbidity and morphology of ulcer were evaluated.The pathological changes of cornea were observed by HE staining.LC3 B and lysosome were detected by co-localization of autophagic double-labeled virus.The expression of ATG5 and P62 were observed by fluorescence microscope.Western blot was used to detect the expression of autophagic protein LC3 B,P62,ATG5,IL-1 and caspase3.Result1.The results of gene chip indicated that miR-665-3p was up-regulated in Fungal Keratitis,and was confirmed by q PCR(P<0.05).Several bioinformatics suggest that ATG5 is one of the targets for MIR-665-3P.Western blot showed that MIR-665-3P could negatively regulate ATG5(P<0.05).Luciferase reporter system confirmed the direct binding site between miR-665-3p and 3’UTR of ATG5(P<0.001).Compared with the control group,the corneal autophagosomes in the experimental group were larger and contained more damaged organelles.Western blot showed that the expression of LC3B-Ⅱ/Ⅰ protein was significantly decreased at 1-5 days,while the expression of P62 protein was increased at 5 days,which indicated that autophagy flux was impaired in Fungal Keratitis(P<0.05).2.Cell experimentWestern blot showed that LC3B-Ⅱ/Ⅰ and P62 protein in CQ group increased significantly,LC3B-Ⅱ/Ⅰ protein increased and P62 protein decreased in Rapa group and ant-665 group,LC3B-Ⅱ/Ⅰ protein decreased and P62 protein increased in miR-665group(P<0.05).The results showed that the yellow or red spots were less in CQ group and miR-665 group than in control group,while the yellow or red spots were more in Rapa group and Ant-665 group than in control group.The results of immunofluorescence showed that the fluorescence intensity of ATG5 protein was decreased in CQ group and miR-665 group,increased in Ant-665 group and Rapa Group.The fluorescence intensity of P62 protein in CQ group and miR-665 group increased,but that in Rapa group and Ant-665 group decreased.3.Animal experimentCompared with the control group,CQ Group,Rapa Group and Ant-665 group had lower corneal edema,turbidity and turbid area,and lower clinical score,while miR-665 group had more serious pathological damage,the degree of Edema,the degree of turbidity and the area of turbidity were more serious than those in the control group,and the clinical score was higher(n = 5).Compared with the control group,the corneal integrity of CQ group and Rapa group was damaged,and there were fewer vacuoles and inflammatory cells in the Stroma,but there were a few vacuoles in the corneal epithelium in Ant-665 group,in miR-665 group,the whole epithelial layer was damaged,the stroma cells were disordered,and there were a lot of inflammatory cells infiltrating the whole layer.The co-localization of the two viruses showed that the yellow fluorescent band polymerization was weakened in CQ group and miR-665 group,but increased in Ant-665 group and Rapa group.The expression of P62 protein was increased in CQ group and miR-665 group,but decreased in Rapa group and Ant-665 group.The fluorescence of ATG5 protein decreased in CQ and miR-665 groups,but increased in Rapa and Ant-665 groups.Western blot showed that CQ group and miR-665 group increased LC3B-Ⅱ/Ⅰ,P62 protein expression and decreased ATG5 expression compared with control group.The expression of P62 protein decreased in Rapa group and Ant-665 group,while the expression of LC3B-Ⅱ/Ⅰ and ATG5 protein increased.The expression of IL-1 and CASPASE3 protein decreased in CQ,Rapa Group and Ant-665 group,however,the expression of IL-1 and CASPASE3 increased in miR-665 group(P<0.05).Conclusion1.Autophagy flux is impaired in Fungal keratitis and miR-665-3p negatively regulates the expression of ATG5.2.MIR-665-3P is involved in autophagic regulation of Fungal Keratitis by targeting ATG5,and inhibition of miR-665-3p expression can enhance autophagic activity of Fungal Keratitis and reduce inflammatory reaction and apoptosis. |
PDF Full Text Request