| Porcine hemagglutinating encephalomyelitis(PHE)is a highly contacted and acutely transmitted disease caused by porcine hemagglutinating encephalomyelitis virus(PHEV).Clinically,the disease occurs mostly in piglets within three weeks and often exhibits two different clinical symptoms..Vomiting failure type: vomiting,diarrhea,failure and other symptoms in sick piglets;encephalomyelitis type: sick piglets have obvious symptoms such as nerve tremor and scream,.The mortality rate of sick piglets can reach 100%.At present,there is no effective preventive and therapeutic measures for porcine hemagglutinating encephalomyelitis.Once the outbreak will bring huge economic losses to the pig industry.PHEV mainly attacks the host nervous system,but the mechanism induced by nerve injury is unclear.Long noncoding RNA(LncRNA)is an RNA molecule that has no coding ability or has little coding ability and is widely present in eukaryotes.Studies have found that a variety of viral infection processes are accompanied by differential expression of LncRNA,such as hepatitis B virus,hepatitis C virus,chicken Marek virus and cytomegalovirus,etc.Whether PHEV infection causes differential expression of LncRNA in the host and its role in the pathogenesis of the virus has not been reported.Therefore,the mechanism of PHEV-induced neuronal damage from the perspective of LncRNA will help to elucidate the pathogenesis of porcine hematologic encephalomyelitis.In this experiment,high-throughput sequencing technology was used to study the expression of LncRNA in the cerebral cortex during brain injury induced by PHEV based on the PHEV-infected mouse model.Compared with the control group,the expression profile of LncRNA in the cerebral cortex of PHEV-infected mice changed significantly.1407 differentially expressed mRNAs were screened,including 1270 mRNAs up-regulated and 137 mRNAs down-regulated.110 differentially expressed LncRNA,including 83 LncRNAs up-regulated and 27 LncRNAs down-regulated.The LncTar software was used to predict the LncRNA target gene based on the principle of sequence complementation.Volcano maps were drawn for differential mRNA and differential LncRNA,respectively,and the expression levels and expression folds of differential mRNA were higher than those of differential LncRNA.GO analysis was used to complete differential mRNA and LncRNA from three parts: biological process,molecular function and cell composition.Functional annotation;functional annotation of differential mRNA and LncRNA from genetic information,environmental information,organic systems,cellular processes,metabolism,etc by KEGG analysis.The gene structure was compared with the length of the differential mRNA and LncRNA,the number of exons and the length of the ORF,which provided experimental basis for the subsequent mining of LncRNA function.Differentially expressed mRNA and LncRNA were extracted and their expression at the cellular and tissue levels was examined by qPCR to test the reliability of high-throughput sequencing.The qPCR results were consistent with the sequencing results,confirming that the high-throughput sequencing results were true and reliable.Two LncRNAs with significant up-regulation of NONMMUT006579.2 and NONMMUT131476.1 were selected from the database as the research object.The expression of NONMMUT006579.2 and NONMMUT131476.1 in the process of PHEV-infected host was detected by qPCR.The results showed that the expression levels of NONMMUT006579.2 and NONMMUT131476.1 were significantly up-regulated compared with the control group,suggesting that these two LncRNAs may be involved in the host process of PHEV infection.To verify the above hypothesis,N2 a cells were transfected with NONMMUT006579.2 siRNA or overexpression vector.After inoculation with PHEV,qRCR results showed that NONMMUT006579.2 RNAi or overexpression vector had no effect on PHEV replication;whereas N2 a cells were transfected with NONMMUT006579.2 siRNA,qPCR and Western blotting methods were used to detect the effect of PHEV proliferation.The results showed that the amount of PHEV replication increased when NONMMUT131476.1 expression was decreased,ie NONMMUT131476.1 negatively regulated viral replication.LncRNA exerts biological regulation by regulating differential expression of related target genes.To further study the regulation of differential LncRNA(NONMMUT006579.2 and NONMMUT131476.1),this experiment uses LncTar software to target genes that may cause brain damage LncRNA.Predicting and docking with high-throughput sequencing results.These target genes were found to be involved in biological processes such as ecological behavior,endocytosis,genetic information,physiological metabolism,cellular processes,and immune responses,such as the Washc4 gene associated with the endocytic pathway.It is the cis target gene of NONMMUT006579.2,and the Nlrc5 associated with innate immunity may be the NONMMUT131476.1 cis target gene.To further investigate whether the expression of differential LncRNA(NONMMUT006579.2 and NONMMUT131476.1)affects the expression of Washc4 and Nlrc5 during PHEV infection.N2 a cells were transfected with NONMMUT006579.2 overexpression vector or siRNA,inoculated with PHEV,and detected by qPCR.The results showed that Washc4 expression and the expression of NONMMUT006579.2 showed a positive correlation;while transfected with NONMMUT131476.1 siRNA and inoculated with PHEV,the results showed that the expression of Nlrc5 was positively correlated with the expression of NONMMUT131476.1.It is suggested that Washc4 is the target gene of NONMMUT006579.2 and Nlrc5 is the target gene of NONMMUT131476.1.In summary,this study completed the screening of LncRNA in the host cerebral cortex after PHEV infection and the interaction analysis between differential LncRNA and differential mRNA.PHEV infection caused up-regulation of host NONMMUT006579.2 and NONMMUT131476.1,while up-regulated expression of NONMMUT006579.2 and NONMMUT131476.1 positively regulated the expression of Washc4 and Nlrc5,respectively,while NONMMUT131476.1 negatively regulated PHEV replication.This study data will support the role of LncRNA in PHEV infection and provide a theoretical basis for treatment options against PHEV infection. |
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