The Regulation And Mechanism Analysis Of APN On The Porcine Epidemic Diarrhea Virus Resistance

Porcine epidemic diarrhea(PED)caused by porcine epidemic diarrhea virus(PEDV)is a highly contagious intestinal infectious disease,which characterized by vomiting,diarrhea and dehydration.Pigs of all ages are susceptible to PEDV,especially for suckling pigs,with a 90%mortality,causing enormous economic losses.Recently,the variant PEDV strains cannot effectively immunize the existing vaccines.Therefore,the effective efforts should be devoted towards the isolation and identification of functional resistance genes against PEDV,thereby genetically preventing and controlling the PED.It has been shown that APN gene is not a key receptor for PEDV to invade host and proliferate.Interestingly,APN gene expression is closely related to the PEDV infection.However,the specific molecular regulatory mechanism remains obscure.Hence,the APN gene expression in the porcine small intestinal epithelial cells(IPEC-J2)infected with PEDV at different time was firstly detected.The SNPs and methylation level in the APN promoter were identified and analyzed to reveal the molecular mechanism of APN gene expression.Additionally,APN knockout cell was constructed to investigate the effect on the cell cycle,cell proliferation,and susceptibility of IPEC-J2 cells to PEDV.Eventually,RNA-seq was performed to explore gene expression changes in APN gene knockout and wild type cells infected with PEDV for 12h,thereby screening the key candidate genes.The results of this study were shown as follows:1.Differential expression of APN gene in IPEC-J2 cells infected with PEDV at different time(0,2,4,6,12,and 24 h)were analyzed using qRT-PCR.Our study demonstrated that APN gene expression was significantly up-regulated in IPEC-J2 infected with PEDV for 4 h(P<0.01),and continued to reach the highest level at 12 h,significantly down-regulated at 24 h(P<0.01).The APN gene knockout IPEC-J2 cell was successfully constructed using CRISPR/Cas9.Differential analysis of cell cycle and cell proliferation on the APN knockout and wildtype cells were performed.Concurrently,the expression of related genes involved in regulating cell cycle were evaluated by qRT-PCR.The observations indicated that the cell viability of APN knockout IPEC-J2 cells was significantly down-regulated at 24h,48h and 72h than that of wildtype(P<0.01).Moreover,the population of IPEC-J2 cells in G1 phase was significantly increased(P<0.01),and the expression of Cyclin D1,Cyclin E1,CDK4,CDK2,and p21,served as the critical factor for the G1 phase regulation,is significantly elevated in the APN knockout cells compared with that in the wildtype(P<0.01).Nevertheless,the population of IPEC-J2 cells in S phase was dramatically reduced(P<0.01).Our findings also showed that APN gene knockout induced a significant decrease in PEDV copies of IPEC-J2 cells infected with PEDV at different time(4,12,and 24 h)(P<0.01).Likewise,the comparison of the susceptibility of IPEC-J2 to PEDV between APN knockout and wildtype cells was performed by indirect immunofluorescence.The results displayed that APN gene knockout reduced the infection capacity of the PEDV.2.The porcine APN gene core promoter was predicted by bioinformatics software,and SNPs located in this region were screened to explore the impact on the promoter activity.The results displayed that three possible core promoter regions were detected,and mutations located in rs 322147864(G/A)and rs326030589(G/A)significantly increased the APN gene promoter activity(P<0.01).In addition,BSP sequencing was used to assess the methylation level of APN gene promoter region in IPEC-J2 cells infected with or without PEDV.The correlation between the methylation level of some sites and the APN gene expression was further analyzed.It was found that the CpG islands with 350 bp length in the APN gene promoter upstream region contained 18 CpG sites.The results indicated that no significant difference was observed on the methylation level of the APN gene promoter between the infected and control group,and the methylation level of the CpG1 was positively correlated with the APN mRNA expression(P<0.01)3.We analyzed the differential gene profile of IPEC-J2 cells and APN knockout cells infected with PEDV by transcriptome sequencing.A total of 3943 genes were identified that were differentially expressed between the APN knockout and wildtype groups(P<0.05),among which the expression of 3121 genes was up-regulated and that of 822 genes was down-regulated.The Gene Ontology analysis indicated that the molecular function of the differential expressed genes was primarily related to receptor activity,molecular transduction and signal receptors,and that the biological functions in which the DEGs are involved included stimulation response,signal transduction,biological quality regulation and multicellular biological processes.Among the disease-related pathways such as cAMP signaling pathway,protein digestion and absorption,Staphylococcus aureus infection,cytokine-cytokine receptor interaction,inflammatory mediator regulation of TRP channels,Calcium signaling pathway,cell adhesion molecules,and leukocyte transendothelial migration were enriched for these DEGs.Furthermore,important candidate genes including CLDN8,DPPA2,FBP1,GTSF1,LAMA1,RGN,CD177,KRT4,LRRC7,and MUC13 were screened based on gene expression fold changes.The qRT-PCR was conducted to verify the transcriptomic analysis results.We confirmed that these genes expression trends were consistent with transcriptome sequencing results.In this study,we further analyzed the impact of APN gene expression on the PEDV resistance and the specific regulatory mechanism.Our study highlighted that higher expression APN gene may induce an increase susceptibility of IPEC-J2 cells to PEDV.The SNPs located in the promoter region significantly elevated the APN gene expression,which may be closely related to the PEDV resistance.In addition,transcriptome analysis preliminarily screened the pathways and candidate genes that closely related to APN,which may play a crucial role in the regulation of PEDV resistance.These findings provide novel insights for detecting the regulatory mechanism of APN gene expression against PEDV and the application of disease resistance breeding.