Expression Of LncRNA Regulating NF-?B Signaling Pathway In PEDV Infected Cells

Porcine epidemic diarrhea is an acute intestinal infectious disease caused by Porcine Epidemic Diarrhea Virus(PEDV).It mainly causes watery diarrhea in newborn piglets within one week of age,accompanied by vomiting,malaise,loss of appetite,and severe dehydration.The death caused great losses to the animal husbandry.Inflammatory reaction in the small intestine is the main cause of diarrhea and dehydration in piglets caused by PEDV infection.This reaction mainly plays a role by activating NF-?B signaling pathway to produce inflammatory cytokines.The regulation of NF-?B signaling pathway includes not only the expression level of protein molecules and post-translational modification,but also the expression and function of mi RNA and lnc RNA at the transcription level.However,whether lnc RNA was involved in the regulation of inflammation during PEDV infection is unknow.In this study,the next-generation transcriptome sequencing technology was used to analyze the expression level of lnc RNA in PEDV-infected pig small intestine and Vero cell samples,it focuse on screening differentially expressed lnc RNA related to the NF-?B pathway.The expression vector was eukaryoticly expressed in Vero cells,analyzing the changes in PEDV replication level after over-expressing lnc RNA.The study initially analyzes the mechanism of lnc RNA in the process of PEDV infection,providing a basis for the study of the pathogenic mechanism of swine epidemic diarrhea.1.Transcriptome sequencing analysis of PEDV-infected piglet small intestine samples andVero cell samplesTranscriptome sequencing is an effective and comprehensive new molecular research method.In this study,the PEDV-YJH strains isolated in the laboratory were used to infect newborn piglets and Vero cell lines,and samples were collected and sent to biological companies for sequencing.Cell sequencing results showed that when PEDV was infected at 0h,the PEDV group had 70 lnc RNA expressions up-regulated and 31 lnc RNA expression down-regulated compared to the MOCK group;at 24 h,the PEDV group had 40 lnc RNAs up-regulated and 54 down-regulated;at 48 h,there were 34 lnc RNAs was up-regulated and 43 down-regulated.The m RNA differential expression profile and lnc RNA differential expression profile were constructed based on the piglet sample sequencing results.Among them,167 genes were up-regulated and 169 genes were down-regulated in the m RNA expression profile,and lnc RNA expression profile was relatively small,containing 47 up-regulated lnc RNA and 62 down-regulate lnc RNA.19 pairs of lnc RNA related to the NF-?B pathway and differentially expressed were selected from the two sets of sequencing results.After screening,the fluorescent quantitative PCR was used to verify.The results showed that the lnc RNA 2694.1 and lnc RNA 3977.1 in the cell samples were infected in the cells.The expression level was significantly provided at 48 h.The expression level of lnc RNA 18325 increased at 24 h and was inhibited at 48 h.At the same time,in piglet samples and cell samples,lnc RNA11810.9 was found to have differential expression of its targeted SUDS3,showing an upward trend.The above results are consistent with the transcriptome sequencing results.2.Analysis on the mechanism of selected lnc RNA and SUDS3 on PEDV infected cellsIn order to further investigate whether lnc RNA has a regulatory effect on the replication level of PEDV,this study constructeslnc RNA 2694.1,lnc RNA 3977.1,lnc RNA 18325 and SUDS3 vectors for eukaryotic expression in Vero cells,and then inoculate 0.6 MOI of PEDV for fluorescence quantification PCR detection of the expression of three kinds of lnc RNA and SUDS3.At the same time we detecte the PEDV viral nucleic acid content in the supernatant.The results showed that: the expression levels of lnc RNA 2694.1,lnc RNA 3977.1,lnc RNA 18325 and SUDS3 increased significantly,but after PEDV infection,the three lnc RNA and SUDS3 The expression level of SUDS3 was inhibited to varying degrees;CPE observation results showed that after PEDV infection,CPE appeared earlier and the disease changed significantly compared with the non-infected virus group;the virus copy number in the cell supernatant of each group was significantly more than that of the uninfected group.The above results indicate that NF-?B-related lnc RNA 2694.1,lnc RNA 3977.1,lnc RNA 18325 and SUDS3 are involved in the regulation of PEDV replication levels,laying a good foundation for further exploration of the inflammatory response mechanism caused by PEDV replication.