Analysis Of The Transcriptional Profiles And The Role Of Mir-204-3p Targeting KRT16 In Tree Shrew Fungal Keratitis

Fungal keratitis is an corneal disease with high rate of blindness caused by pathogenic fungi,Fusarium solani is the most common pathogenic fungus.At present,the effect of drug treatment is not ideal since there's a lack of low toxicity and high efficiency antifungal drugs.The lack of corneal donor and high recurrence rate of infection after operation make the effect of surgical treatment of fungal keratitis unsatisfactory.fungal keratitis has become one of the most intractable ophthalmic diseases in infectious keratitis,there is no effective method to cure fungal keratitis.Animal models are an important means to investigate the pathogenesis of fungal keratitis and develop antifungal drugs.The corneal structure of rodents and rabbits is different from that of humans,which limits its application in study of human corneal diseases.The corneal structure of tree shrews is very similar to that of human,and the corneal symptoms in the early stage of infection with fusarium solanum are similar to those of human fungal keratitis.Revealing the regulatory mechanism of fungal keratitis symptoms in tree shrew is beneficial to study the pathogenesis of human fungal keratitis and provide basic data for the development of antifungal drug.MethodsIn this study,the tree shrew fungal keratitis model was used to study the corneal expression profiles of mRNA,miRNA,circRNA and LncRNA in different disease courses of tree shrew fungal keratitis(7 days,14 days and 30 days after Fusarium solani infection)by high-throughput sequencing technology.The differentially expressed genes were analyzed and network regulatory pathway were constructed in different infection periods of tree shrew fungal keratitis,including:(1)The associations of miRNA-mRNA?circRNA-mRNA?LncRNA-mRNA were analyzed for the main metabolic pathways involved in miRNA,circRNA and LncRNA.(2)The regulatory networks of LncRNA-mRNA-miRNA?circRNA-mRNA-miRNA were constructed to analyze the main response regulators of decreased fungal keratitis symptoms in tree shrews.At the same time,miRNA and its target mRNA were screened under different course of fungal keratitis in tree shrew,and the target relationship between miRNA and mRNA was studied by luciferase reporter gene.miRNA agomir was injected into corneal of tree shrew with fungal keratitis to investigate the role of miRNA and its target mRNA in the pathogenesis of tree shrew fungal keratitis.ResultsAfter 7 days of infection,78 mRNAs(62 upregulated and 16 down-regulated),70 miRNAs(31 upregulated and 39 downregulated),59 circRNAs(32 upregulated and 27 downregulated)and 141 lncRNAs(133 upregulated and 8 downregulated)were significantly differentially expressed.After 14 days of infection,56 mRNAs(54 upregulated,2 downregulated)and 63 miRNAs(19 upregulated,44 downregulated),14 circRNAs(8 upregulated and 6 downregulated)and 31 lncRNAs(22 upregulated and 9 down-regulated)were significantly differentially expressed.After 30 days of infection,41 mRNAs(37 upregulated and 4 downregulated)and 20 miRNAs(18 upregulated and 2 downregulated).6 circRNAs(5 upregulated and 1 downregulated)and 36 lncRNAs(24 upregulated and 12 down-regulated)were significantly differentially expressed.There are 29 mRNAs and 14 miRNAs that were significantly differentially expressed after 7 days,14 days and 30 days of infection.The significantly differentially expressed genes were mostly enriched in the biological functions of stress and immune response,and the PI3K-Akt,NF-?B and Jak-STAT signaling pathways.There were 13 mRNAs(all upregulated)significant different expressed after 7-day,14-day and 30-day infection.These mRNAs mainly enriched in the PI3K-Akt signaling pathway.3 miRNAs and 7 lncRNAs that different expressed have targeted relationships with these genes.In the early days of infection(after 7-day and 14-day of infection),23 significantly differentially expressed mRNAs(16 upregulated,7 downregulated)mainly enriched in the PI3K-Akt and Jak-STAT signaling pathways.11 miRNAs and 2 lncRNAs that different expressed have targeted relationships with these genes.At the late stage of infection(after 14-day and 30-day of infection),10 significantly differentially expressed mRNAs(9 upregulated,1 downregulated)mainly enriched in the MRS A infection related pathway(KEGG pathway:tup05150).7 different expressed miRNAs have targeted relationships with these genes.qPCR was used to detect the expression of ALPL,TREML1,PDE1B,KLK7 genes and has-miR-214-3p,has-miR-149-5p in tree shrew corneal after 7-day,14-day and 30-day infection with fusillaria solanum.The qPCR results were consistent with the results of RNA sequencing.There were 4 mRNAs,7 miRNAs and 37 lncRNAs with significantly different expressions were used to construct 49 lncRNA-miRNA-mRNA regulatory networks,25 miRNAs,38 circRNAs and 62 mRNAs with significant differential expression were used to construct 188 circRNA-miRNA-mRNA regulatory networks.After 7 days,14 days and 30 days of Fusarium solani infection,miR-204-3p and KRT16 genes were differentially expressed.Luciferase reporter genes showed that miR-204-3p binds to 3'UTR region of KRT16 gene.The results of in vivo experiments showed that the expression level of mir-204-3p in tree shrew corneal with miR-204-3p injection was significantly higher than that of control eye after 7 and 14 days injection,and the expression level of KRT16 protein in corneal with miR-204-3p injection was lower than that of control eye.Corneal histopathological examination showed that,the symptoms of fungal keratitis were decreased in corneal with miR-204-3p injection,and corneal wound heal was obvious.ConclusionIn this study,high-throughput sequencing was used to investigate the molecular mechanism of decreased fungal keratitis symptoms in a tree shrew.Key differentially expressed genes including KRT16,TREML1,LPS,CCL2,CCR2,CCL7,CCR7 and IL-1?,and important miRNA including miR-214-3p,miR-149-5p and miR-204-3p were obtained by analyzing gene expression characteristics between the infected eyes and self-uninfected eyes,and important signaling pathways were obtained,including PI3K-Akt?NF-k?B and Jak-STAT signaling pathways.In addition,this study demonstrated that miR-204-3p promote corneal wound healing of tree shrew fungal keratitis by targeting KRT16.In this study,we constructed ceRNA networks to elucidate the interaction among different expressed genes after fungal solani infection,which will provide an important basis for the research on the pathogenesis of human fungal keratitis and the selection of antifungal drug targets.