Study On Porcine Epidemic Diarrhea Virus Replication Regulated By LncRNA-9560 And LncRNA-9606

Long non-coding RNAs(lncRNAs)are kind of RNA molecules with multiple functions and participate in the regulation of biological events.Recent research results demonstrated that there was a phenomenon of differential expression of lncRNA in virus-infected tissues or cells.Currently,there are more studies focusing on human lncRNA but few studies on animal lncRNA.The purpose of this study is to analyze the lncRNA expression profile in IPEC-J2 cells infected with porcine epidemic diarrhea virus(PEDV)and the regulatory function of differentially expressed lncRNA on with PEDV-infected cells.Firstly,in order to investigate whether PEDV infection affects the expression level of host lncRNA,a transcriptome sequencing was employed to analyze the lncRNA expression in IPEC-J2 cells infected with PEDV.The results illustrated that the PEDV induced differential expression of lncRNA in cells,showing different changes with the time of viral infection.Functional enrichment of differentially expressed lncRNA was performed by using GO and KEGG.The results showed that the signal pathways enriched by the host lncRNA varied at different time points post PEDV infection,indicating that the main biological processes were also different at different stages.The target gene prediction analysis revealed that lncRNA-9560 and lncRNA-9606 target the same transcript,whose coding protein was closely related to IgA production.Subsequently,the basic characteristics of these two lncRNAs were further analyzed.The results showed that the expression levels of lncRNA-9560 and lncRNA-9606 in the cells increased with the time duration upon PEDV infection.The nuclear mass separation results demonstrated that both lncRNAs were distributed in the nucleus and cytoplasm,but mainly in the nucleus.To verify that the lncRNA-9560 and lncRNA-9606 were noncoding RNA,the wild type and truncated recombinant plasmids of these lncRNAs fused with His-tag at the 3' terminus were constructed and transfected into HEK-293 T cells.Western-blotting results showed that both lncRNA-9560 and lncRNA-9606 were unable to encode proteins.To investigate whether lncRNA-9560 and lncRNA-9606 affect PEDV replication,quantitative PCR and western-blotting were used to analyze the PEDV genomic copies and the expression level of PEDV-N protein in lncRNA-overexpressed LLC-PK1 cells.The results presented that both lncRNA-9560 and lncRNA-9606 inhibited PEDV replication.To further investigate whether lncRNA-9560 and lncRNA-9606 were involved in cytokine expression,the quantitative PCR was used to detect their transcription level of cytokines in lncRNA overexpressed or knocked-down IPEC-J2 cells.The results illustrated that overexpression of lncRNA-9606 promoted the transcription of GM-CSF,and vice versa.However,the lncRNA-9560 failed to regulate TNF-? and GM-CSF expression at the transcription level.In summary,this study systemetically analyzed the expression profile of lncRNA in PEDV-infected IPEC-J2 cells,and discovered that differentially expressed lncRNA-9560 and lncRNA-9606 inhibited PEDV replication.Meanwhile,lncRNA-9606 promoted GM-CSF expression at transcription level.This research expanded the knowledge about regulation of viral replication by lncRNAs,and provided theoretical basis for lncRNA-based drug discovery for PEDV treatment.