Identification And Functional Verification Of Protein Coding Genes And LncRNAs In Trichophyton Mentagrophytes–induced Rabbit Dermatophytosis

Dermatophytosis is a common disease in the rabbit.Dermatophytosis is mainly caused by filamentous fungi,which can specially invade the stratum corneum of hosts and its appendages.Dermatophytosis in the rabbit most often is reported to be caused by Trichophyton mentagrophytes(T.mentagrophytes).At present,the mechanism of the rabbit's response to the dermatophytes is not clear.Therefore,this study intends to identify the protein coding genes and lncRNAs related to the pathogenesis of rabbit dermatophytosis by RNA-seq technique,and to explore the function of key protein coding genes in the pathogenesis of rabbit dermatophytosis at the cell level.Our study constructed rabbit dematophytosis model by injecting T.mentagrophytes on rabbit dosal skin.Next,RNA-seq and bioinformatics technology were adopted to identify protein coding genes and lncRNAs related to the pathogenesis of rabbit dermatophytosis.At last,we constructed rabbit keratinocyte inflammatory cell model by?-glucan stimulation.In this model,we analyzed the role of JAK2-STAT3 signaling pathway on the secretion of CXCL8,CXCL11 and IL-1?using RNAi technology and overexpression technology.The main results were as follows:(1)After infecting rabbit skin with 1.0×10~6 CFU/mL T.mentagrophytes,all infected rabbits showed obvious dermatophytosis-like phenotypes,including redness,incrustation,scaling and ulceration.GMS stain indicated the infected skin was invaded by T.mentagrophytes.H.E.stain showed that the infected skin appeared local parakeratosis,stratum spinosum hypertrophy,and a large number of inflammatory cells infiltration in papillary layer and reticular layer.The above results showed that the rabbit dematophytosis model was successfully constructed.(2)Following RNA-seq and bioinformatic analysis,we identified 19,720 protein coding genes from 12 RNA sequencing libraries,including 10,620 known and 9100 novel protein coding genes.Of 292 differentially expressed genes,227 genes(q<0.05)were induced while 65 genes were repressed in TM relative to NS.We also identified significant induction of key dermatophytosis-related genes,which involved in fungal recognition,inflammation response,and nutritional immunity.GO functional enrichment analysis indicated that highly represented GO terms included‘Immune response',‘Response to external stimulus',‘Inflammatory response',‘Defense response',‘Response to stress'.KEGG pathway analysis revealed that‘Cytokine-cytokine receptor interaction',‘Toll-like receptor signaling pathway',‘Natural killer cell mediated cytotoxicity',‘Chemokine signaling pathway',‘TNF signaling pathway',‘NF-kappa B signaling pathway'were significantly(q<0.05)enriched.(3)We identified 5,883 lncRNAs in 12 strand-specific RNA-seq libraries and found64 differentially expressed lncRNAs(q<0.05)in TM relative to NS.Rabbit lncRNAs were generally smaller in size,fewer in exon numbers and longer in exon size compared to protein coding genes.Co-expression analysis revealed that 107 pairs between 32 DE lncRNAs and 96 protein coding genes showed a highly correlated expression(|r|>0.8).Gene ontology enrichment analysis of the protein coding genes associated with the 32 DE lncRNAs revealed‘Keratin filament',‘Antigen binding',‘MHC protein complex',‘Antigen processing and presentation',‘Interleukin-1 receptor binding'and‘Immune response'as some of the significant biological processes.Pathway analysis indicated that the co-expressed protein coding genes were mainly enriched in‘Antigen processing and presentation',‘Endocytosis',‘Jak-STAT signaling pathway',and‘Cytokine-cytokine receptor interaction'.Our results demonstrated that lncRNAs might be involved in dermatophytosis pathogenesis by co-expression with protein coding genes.MiRPara analysis of the lncRNAs revealed 173 lncRNAs with precursor sequences for 9561probable novel miRNAs.And 641 of these miRNAs targeted 10 protein coding genes.GO enrichment analysis of these protein coding genes showed that they were enriched in some dermatophytosis related terms,such as immune system process,response to stress and response to stimulus.These results indicated that lncRNAs might be responsible for host responses against T.mentagrophytes by being processed into miRNAs.(4)In this experiment,different concentrations of?-glucan simulating the fungal antigen were used to infect the rabbit KC.The inhibition rate of 20 ug/mL?-glucan on KC is closest to 10%,which could be used as the optimal concentration on KC.After infecting KC with?-glucan,the secretion of CXCL8,CXCL11 and IL-1?in KC was significantly higher than that in the control group,indicating that the?-glucan-induced KC inflammatory cell model was successfully constructed.In the KC inflammatory cell model,?-glucan stimulation could induce the expression of JAK2 mRNA and p-STAT3.Silencing the expression of JAK2 could inhibit p-STAT3 expression and IL-1?secretion induced by?-glucan.And over expression of JAK2 could further promote the induction effect of?-glucan on p-STAT3 expression and IL-1?production.These results suggested that?-glucan could induce the secretion of IL-1?by activating the JAK2/STAT3 signaling pathway and indicated that IL-1?induced by JAK2/STAT3 signaling pathway in KC was involved in the T.mentagrophytes induced inflammation of the rabbit dorsal skin.This study provideed a reference for further revealing the molecular mechanisms and resistance breeding of rabbit dermatophytosis by identifying the protein coding genes and lncRNAs involved in the pathogenesis of rabbit dermatophytosis and investigating the role of JAK2/STAT3 signaling pathway and some protein coding genes on?-glucan induced KC inflammatory cell model.