| OBJECTIVE: To investigate the role and mechanism of miR-129-5p inhibition by Atg14-mediated autophagy in fungal keratitis.method: 1.Using bioinformatics software Target Scan(http://www.targetscan.org/)to predict and analyze the target gene of miR-129-5p,and screen for Atg14 as the target gene of miR-129-5p,through luciferase(Luciferase)Two gene validations were performed in the reporter gene detection technique and the mouse fungal keratitis model.2.Mouse corneal stromal cells were cultured in vitro and divided into groups: normal uninfected group;normal infection group(control group);autophagy group(3-MA group);autophagy group(Rapa group);miR-129-5p inhibitor group(antagomir group);miR-129-5p mimic group(agomir group);miR-129-5p negative control group(NTC group).After the fungal spores were infected with mouse corneal stromal cells(multiplicity of infection=5),the expression of miR-129-5p was detected by RT-PCR at 0,3,6,12 and 24 h;Western-Blot was used to detect Atg14.The expressions of Beclin1 and LC3 B proteins were different.The fluorescence signal intensity of Atg14,Beclin1 and LC3 B proteins in each group was observed by laser confocal microscopy.The difference of LC3 B protein and autophagosome lysosome was observed to detect the difference of autophagic flow in each group.The cell scratch test was used to detect the difference of cell migration ability of each group;CCK-8 was used to detect the difference of cell viability in each group.3.Animal experiment and grouping: 6-8 weeks old BALB/C male mice were randomly divided into 7 groups and given subconjunctival administration: normal uninfected;normal infection group(control group,n=3);inhibition Autophagy group(3-MA group,n=3);activated autophagy group(Rapa group,n=3);miR-129-5p antagomir group(n=3);miR-129-5p agomir group(n= 3);miR-129-5p NC group(NTC group,n=3).After 1 hour of administration,the model of Fusarium solani keratitis was successfully established by modified surface mirror method.After the model was established,the corneal slit lamp of the corneal infection fungus was observed and recorded at five time points of 1,3,5,7 and 10 days.Characteristics of lower lesions;clinical scoring of corneal opacity area,density,and regularity of lesion surface;corneal bacterial load test to detect differences in fungal clearance in each group;RT-PCR detection of fungal keratitis lesion progression during miR-129-The expression of Atg14,Beclin1 and LC3 B protein in each group was detected by Western-Blot.The fluorescence intensity of Atg14,Beclin1 and LC3 B proteins in the cornea of each group were observed by immunofluorescence staining.The IL-6 and IL-in ELISA were detected by ELISA.The expression levels of 1? in each group at the time points of infection on days 1,3,5,and 7.result: First,the luciferase reporter gene results showed that miR-129-5p significantly down-regulated the reporter fluorescence of Atg14.When the target site was mutated,the reporter fluorescence recovered,and miR-129-5p did pass the Atg14 3'UTR.In combination with direct regulation of Atg14 expression,Atg14 was the target gene of miR-129-5p;western-blot was used for protein expression detection.The results showed that miR-129-5p antagomiR group was compared with antagomiR NTC group,miR-129-5p significantly promoted Atg14 protein expression(P<0.05);miR-129-5p agomiR group significantly inhibited Atg14 protein expression compared with agomiR NTC group(P<0.05).The above results showed that miR-129-5p is negative for Atg14 protein.Sexual adjustment.The results of RT-PCR of mouse corneal stromal cell model showed that the expression of miR-129-5p was significantly increased in corneal stromal cells at 6h after infection with fungal spores.The results of Western-Blot experiment 6h after infection with fungal spores The expression levels of Atg14,Beclin1 and LC3B in stromal cells were significantly higher in the miR-129-5p antagomir group and Rapa group than in the normal infection group and NTC group,and the expression in the 3-MA group was significantly decreased(P< 0.05);Cell laser confocal results showed that compared with the normal infection group and the NTC group,the fluorescence signals of Atg14,Beclin1 and LC3B proteins in the miR-129-5p antagomir group and the Rapa group were significantly enhanced,and the miR-129-5p agomir group,The fluorescence signal intensity of Atg14,Beclin1 and LC3 B proteins in 3-MA group was significantly weakened.The colocalization of LC3 B protein and autophagosomes suggested that miR-129-5p antagomir group and Rapa group autophagy compared with normal infection group and NTC group.The co-localized fluorescence signal intensity of the flow was significantly stronger than that of the miR-129-5p agomir group and the 3-MA group.The statistical results of cell scratch test after 12 h infection of fungal spores showed that the migration area of cells in miR-129-5p antagomir group and Rapa group was significantly increased compared with normal infection group and NTC group,while miR-129 The-5P agomir group and the 3-MA group were significantly reduced;the cck-8 cell viability assay showed that the cell viability of the miR-129-5p antagomir group and the Rapa group was significantly higher than that of the normal infection group and the NTC group,while miR-129 The cell viability of the-5p agomir group and the 3-MA group was significantly decreased.Experimental results of animal model: Corneal image acquisition and observation of corneal inflammation under slit lamp and its lesion characteristics and clinical score showed that the cornea of miR-129-5p antagomir group and Rapa group were compared with normal infection group and NTC group.The inflammatory reaction is mild,local ulcers and necrosis are less likely to occur,the cornea is not easy to perforate,the course of disease is shortened,and the neovascularization of the cornea is the lightest;the results of corneal bacterial load show that the corneal fungal load in mice shows an overall decline with time.The trend of fungal clearance was the fastest in the miR-129-5p antagomir group and the Rapa group compared with the normal infection group and the NTC group,while the fungal clearance rate was the slowest in the miR-129-5p agomir group and the 3-MA group.P <0.05);RT-PCR results showed that the expression of miR-129-5p was significantly increased in the cornea after the third day of infection.WesternBlot statistics showed that the expression levels of Atg14,Beclin1 and LC3 B in the cornea on the third day of infection were normal.Compared with the NTC group,the expression of miR-129-5P antagomir group and Rapa group was significantly increased and the expression of 3-MA group was significantly decreased(P<0.05).The pathological staining results of tissue frozen sections showed that: normal infection group Compared with the NTC group,the fluorescence signals of Atg14,beclin1,and LC3 B in the miR- 129-5P antagomir group and the Rapa group were significantly enhanced,while the fluorescence signal intensity of the AtR14,beclin1,and LC3 B proteins in the miR-129-5P agomir group and the 3-MA group were significantly weakened.The results of ELISA cytokine assay showed that IL-6 expression was significantly increased in the early stage of infection,and the expression of miR-129-5p antagomir group and Rapa group was the most significant compared with the normal infection group and NTC group(P< 0.05).IL-1? expression was mainly concentrated in the middle and late stages;compared with the normal infection group and the NTC group,the miR-129-5p agomir group and the 3-MA group had the most significant increase(P<0.05).Conclusion: miR-129-5p directly regulates the target gene Atg14;miR-129-5p plays an important role in the development of mouse fungal keratitis by targeting Atg14,regulating the expression of autophagy-related proteins and regulating the inflammatory response.. |
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