Analysis Of MiRNA-mRNA Profiles In PEDV-Infected Swine Testicular Cells And Molecular Mechanisms Of Effect Of MiRNA On PEDV Replication

Since 2010,porcine epidemic diarrhea virus(PEDV)variant virulent strains have brought significant challenges to the development of the pig industry in major pig producing countries.Although vaccines have been developed to prevent PED,PEDV variant strains still cause high mortality in the vaccinated populations.Therefore,it is crucial to investigate the pathogenic mechanism of PEDV variant virulent strains for developing anti-PEDV strategies.This study first screened swine testicular cells(ST cells)as a suitable cell line for studying in vitro infection of PEDV variant virulent strain(YN).small RNA and transcriptome of PEDV variant virulent and attenuated YN strains infected ST cells were performed through high-throughput sequencing technology.Moreover,the differences in pathogenicity and host immune response between PEDV virulent and attenuated strains were explored based on the combined analysis of differentially expressed(DE,p-value<0.05)miRNA-mRNA.We found that the expression of miR-155-5P and miR-128 could be regulated in ST cells infected with PEDV,and both of them could inhibit the replication of YN15 strain by targeting host and viral genes,and thereby regulate different signaling pathways.Furthermore,the roles of their target genes in PEDV infected ST cells were explored.The main research contents were as follows:1.Screening of cell lines infected by PEDV variant strain in vitroThis study compared the proliferation of PEDV variant virulent(YN15)strain in ST cells,Vero cells and IPEC-J2 cells by TCID₅₀,IFA(indirect immunoinfluscent assay)and Western Blot assay.The results showed that YN15 strain could efficiently replicate in ST cells.Moreover,viral titer,and the expression of viral protein could reach a high level.The progress of virus infection could be easily observed,given that the infected cells showed clear cytopathic effect(CPE)after infection under an inverted microscope.YN144 strain was also proved to infect ST cells.Moreover,it met the requirement that ST cells were from pig with a complete signaling pathway.Therefor,ST cells were selected as the infection model for variant PEDVs.2.Analysis of miRNA-mRNA expression profile of ST cells infected by PEDV variant virulent and attenuated strainsThe PEDV variant virulent strain(YN1)was isolated by our laboratory,it was further passaged in vitro and obtained YN15 and YN144 strains.In vivo,it was shown that YN15 strain could cause severe diarrhea,however,the attenuated YN144 strain could induce strong innate immune response and neutralizing antibody response in piglets.To investigate the mechanism of the above phenomenon,small RNA-and m RNA-seq of ST cells infected by PEDV variant virulent strain YN15 and the attenuated strain YN144 were performed by high-throughput sequencing technology.The DE miRNA and m RNA profiles were obtained(p<0.05).The results showed that there were8039,8631 and 3310 DE m RNA,and 36,36 and 22 DE miRNA among YN15 vs control,YN144 vs Control,and YN15 vs YN144 groups,and were confirmed by RT-q PCR and stem-loop RT-q PCR.Then,miRanda was used to predict the targeting relationship between miRNA and m RNA.A total of 14140,15367 and 3771 differentially expressed and negatively correlated miRNA-mRNA interaction pairs were found,and two interaction networks-related to viral pathogenicity and host immune response were constructed by Cytoscape software between YN15 and YN144 strains infected-ST cells,respectively.Bioinformatics analysis showed that YN15 strain had apparent advantages over YN144 strain in focal adhesion,cytoskeleton regulation,endocytosis,and bacterial invasion of epithelial cells.However,YN144 strain had apparent advantages in activating NF-?B,JAK-STAT,T cell receptor and other immune-related signaling pathways.3.miR-155-5p affected replication of PEDV YN15 strainCombined analysis of the DE miRNA-mRNA in YN15 strain infected ST cells showed that some genes related to endoplasmic reticulum(ER)stress were up-regulated.It means that ER stress might be associated with the replication of PEDV in ST cells.It has been found that miR-155-5p mimic could inhibit YN15 strain replication in ST cells through its seed sequence targeting PEDV NSP2 gene and the 3'UTR of e IF2?(eukaryotic initiation factor)gene.miR-155-5p mimic could inhibit the transcription of ATG5 and ATG3 genes related to autophagy,and e IF2?,and promote the transcription of MX1 and MX2 genes related to native immunity,and the transcription of IL18 and MAPK1 genes related to acquired immunity.We also found that e IF2?phosphatase inhibitor(Sal003)could effectively repress YN15 strain replication,and promote the transcriptional level of NFKB1,MX1,MAPK1and other genes related to the host immune response in ST cells.4.miR-128 affected replication of PEDV YN15 strainCombined analysis of the DE miRNA-mRNA of ST cells after infection by PEDV YN15 strain showed that some autophagy-related genes were up-regulated,so,autophagy might be related to PEDV replication in ST cells.We found that miR-128 mimic could inhibit PEDV replication in ST cells by its seed sequence targeting PEDV NSP13 gene.miR-128 mimic could also inhibit the transcription of ATG3,ATG5 and ATG12 genes related to autophagy,and promote the transcription of MX1 and MX2 genes related to native immunity,and IL18 and MAPK1 genes related to acquired immunity.To explore the relationship between autophagy and replication of YN15 strain in ST cells,ATG5 and ATG12 were silenced,then ST cells were infected by YN15 strain.The results showed that silencing ATG5 and ATG12 could inhibit PEDV replication,the transcription of e IF2AK3 and other ER-related genes,and the transcription of MX1,MX2,MAPK1,and other genes in host immune-related signaling pathways.In addition,we found that the CBLB genes was significantly up-regulated in ST cells infected with YN15 strain.CBLB is a T cell receptor suppressor gene,which can inhibit the host immune.In this study,we found that miR-128 seed sequence could target the 3'UTR of CBLB gene and inhibit CBLB gene transcription.Furthermore,overexpression of CBLB could promote the replication of YN15 strain.However,si CBLB could inhibit YN15 replication through up-regulating the transcription of some immune-related genes in ST cells,including NFKB1,and IL18.In summary,miRNA and m RNA differentially expressed profiles of PEDV variant virulent strain and the attenuated strain-infected ST cells,and the miRNA-mRNA interaction networks were established.It revealed that the molecular mechanism of miR-155-5p and miR-128 on inhibiting the replication of PEDV,and providing new insight into the molecular mechanism of host factors on inhibiting PEDV replication and new clues for the molecular design of anti-PEDV drugs.