Alternations Of M~6A-modified Transcript Profiles In Mice Corneal Tissues Infected With Fusarium

Objective:The purpose of this study was to construct and analyze the N6-methyladenosine(m~6A)modified map of fungal keratitis(FK)in mouse model,which provide a new sight for elucidating the pathogenesis of fungal keratitis and aim to provide new ideas and feasible targets for diagnosis and treatment of FK.Methods:The corneas of BALB/C mice were infected with Fusarium solani(F.solani),and those treated by phosphate Buffer solution(PBS)as control.The overall m~6A level was detected via an m~6A RNA methylation assay kit.The expression levels of key m~6A modification-related genes were estimated by PCR.The expression and localization of METTL(methyltransferase like)3,was determined by immunostaining and Western blotting.Immunoprecipitation methylation microarray was used to describe the changes in m~6A modification in F.solani-infected corneal tissue,afterwards,Gene Ontology(GO)function enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)signal pathway enrichment analysis of differential m~6A modified mRNAs were performed.Results:BALB/C mice infected with F.solani showed typical fungal keratitis symptoms such as corneal edema,erosive necrosis and ulcer on the 1st day post-infection,and the clinical score reached the peak on the 5th day post-infection.Compared with the control group,the overall levels of m~6A in corneal increased significantly 5 days after being with F.solani We detected the expression levels of several m~6A-related genes,the results showed that the expression of METTL3 and Methyltransferase-like 14(METTL14)were up-regulated in corneal tissue infected by F.solani,especially METTL3.However,there were no significant differences in the expression levels of other genes such as Fat mass and obesity-associated gene(FTO),AlkB homolog 5(ALKHB5)and Wilms tumor 1-associated protein(WTAP).Western blot and immunofluorescence assay further confirmed the increased expression of METTL3 at protein level in the cornea of infected mice.By comparing the m~6A-modificated profiles between the fungal keratitis group and the control group,and based on the threshold at|log2Fold Change|≥2 and False Discovery Rate(FDR)≤0.05,a total of 1137 genes with significant difference in m~6A modification were screened,of which 780 were hypermethylated and 357 were hypomethylated.Bioinformatics analysis of these hypermethylated mRNAs showed that these mRNAs were mainly enriched in systematic and vascular development(biological process),extracellular region and extracellular space(cellular composition),extracellular matrix structural components and protein binding(molecular function).KEGG pathway analysis showed that these hypermethylated mRNAs were mainly enriched in phosphatei-dylinositol-3-kinase/serine-threonine kinase(PI3K-Akt)signaling pathway,cytokine receptor interaction and(tumor necrosis factor(TNF)signaling pathway.The elevated degree of phosphorylation of Akt and PI3K in the cornea of fungal keratitis mouse models were confirmed by Western blot.Conclusions:In summary,the overall level of m~6A in the cornea after fungal infection showed an upward trend,and METTL3 might be the reason for the higher m~6A levels.To our best knowledge,this was first study to construct the m~6A-modified transcript landscape of fungal keratitis in mouse models,and bioinformatics analysis showed that PI3K-Akt signal pathway might be the promising target in further study.