The Transcriptomic Study Of Fungal Keratitis Model Based On High-throughput Sequencing

Fungal keratitis is a blinding eye disease with a high incidence in the world.However,due to the sensitivity of different strains of fungi to antifungal drugs,there is no effective treatment for this disease.At the same time,we found that the animal body itself has an inherent immune response mechanism to fungal infection,in order to quickly and effectively identify pathogens and resist pathogenic invasion.There are various pattern recognition receptors(PRRs)in different cell populations,such as C-type lectin receptors(CTLRs),Toll-like receptors(TLRs),RIG-1-like receptors and nucleotide-binding oligomerization domains(NLRs)containing Nod protein,which can identify fungal invaders and trigger signal transduction pathways and cell responses to eliminate pathogens.However,previous studies mostly focused on single receptor factors,lacking a systematic analysis of the genetic factors underlying the pathogenesis of fungal keratitis.Therefore,we performed a transcriptomic analysis on the model of fungal keratitis in mice by using high-throughput sequencing technology to obtain the transcriptome data of relevant samples and to screen the differentially expressed gene sets,signaling pathways and expression regulation networks of key factors in the pathogenesis of fungal keratitis in mice through statistical analysis of big data.The experimental contents are as follows:Firstly,we successfully constructed a C57BL/6 mouse model of Aspergillus fumigatus infection of corneal tissue.Thirty-eight mice were fed adaptively for one week and randomly divided into three groups: 1)blank group: 5 mice had no cornea intervention;2)control group(PBS group): 15 mice were injected PBS into the corneal stroma of the right eye without any treatment in the left eye;3)experimental group(S group): 18 mice were injected spore suspension into the corneal stroma of the right eye,and the left eye was not allowed to do anything.All mice were cultured for 5 days.Secondly,PBS1 was selected as the control group,S2 was taken as the cornea sample on the second day of culture,S5 was taken as the experimental sample on the fifth day of culture.After RNA extraction,purification and library establishment,the second generation sequencing(NGS)was used to sequence these libraries based on Illumina Hiq X10 sequencing platform.Paired-end(PE)sequencing was performed.Thirdly,the high-throughput sequencing data of 6G clean data for each group were obtained by strict quality control of sequencing samples and data.The Q20 level and Q30 level of the three groups of samples were all above 95% and 90%.Based on principal component analysis and sample correlation test,there was obvious heterogeneity in the three groups of sequencing samples,and the data quality met the follow-up requirements for transcriptomic analysis.Fourthly,by comparing the gene expression profiles of PBS1 group and S2 group(S2group/PBS1 group),and based on threshold criterion | log2 Fold Change | > 1 and significant P-value < 0.05,we screened 646 differentially expressed genes,including455 up-regulated genes and 191 down-regulated genes,such as the up-regulated gene of Spdef and down-regulated gene of Galnt15;by comparing the gene expression profiles of S5 group and S2 group(S5 group/S2 group),the total number of differentially expressed genes was 739,including 650 up-regulated genes and 89down-regulated genes,such as the up-regulated gene of Marco and down-regulated gene of Ccl4.The results of comparison of significant differentially expressed gene sets at different time points showed that there were three commonly down-regulated genes,including Gldc,Aldh1a1 and Ppp1r3 c genes,and 49 commonly up-regulated genes,such as Casp12 and Il1r2 genes,in both groups.Interestingly,the number of genes up-regulated in group S2/PBS but down-regulated in group S5/S2 was 33,including Il1 b gene;down-regulated in group S2/PBS but up-regulated in group S5/S2,including Wnt5 a gene.Most of the identified genes have been reported in previous studies on fungal ophthalmopathy or immunology.Fifthly,we identified signal transduction pathways associated with the pathogenesis of fungal keratitis in mice based on gene enrichment analysis.The significantly enriched signal transduction related pathways in S2/PBS process include TNF signaling pathway(FDR=2.32E-11),NF-kappa B signaling pathway(FDR=2.66E-05),PI3K-Akt signaling pathway(FDR=3.86E-05),JAK-STAT signaling pathway(FDR=7.70E-05),MAPK signaling pathway(FDR=2.03E-03),Rap1 signaling pathway(FDR=1.46E-02)and HIF-1 signaling pathway(FDR=3.54E-02).In contrast,Wnt signaling pathway(FDR = 3.62E-02),c GMP-PKG signaling pathway(FDR = 4.10E-02)and Hippo signaling pathway(FDR = 4.27E-02)were specifically enriched on the fifth day of fungal infection in mice cornea.In addition,we constructed a Card9-Casp1-Il1 b regulatory network based on interleukin-1beta gene Il1 b as a key target gene locus for gene expression regulation in mice with fungal keratitis to elucidate the pathogenesis of fungal keratitis in mice.The expression pattern of Card9 in the mouse model of fungal keratitis was preliminarily explored.Furthermore,the expression pattern of C-type lectin receptor signaling pathway genes(especially the Card9 gene and inflammatory cytokines)in the mouse model of fungal keratitis was explored.Immunofluorescence(IF)and real-time fluorescence quantitative PCR(q RT-PCR)results showed that the expression of Card9 gene in three groups of samples,namely PBS1 group,S2 group and S5 group,was lower.Furthermore,the expression of Card9 gene showed an upward trend on the second day of fungal infection,but its expression level showed a downward trend on the fifth day of fungal infection.In conclusion,the transcriptomic study of fungal keratitis mouse model based on high throughput sequencing technology provides a theoretical basis and data support for the systematic analysis of the pathogenesis of fungal keratitis,and can provide more target sites for its drug treatment,such as Card9.