Studies On Changes Of MRNA And MicroRNA Expression Profile In Mice Cerebral Cortex Infected With PHEV And The Mechanism Of PHEV-indued Cell Apoptosis

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a positive-sensenon-segmented,single-stranded RNA virus that belongs to the Betacoronavirus family.PHEV causes encephalomyelitis, or vomiting and wasting disease in suckling piglets.When piglets under3weeks old are infected with PHEV, their mortality rate is higher,up to100%. Procine hemagglutinating encephalomyelitis virus (PHEV) causes acutedeadly encephalomyelitis in natural host piglets, yet its pathogenesis remains poorlyunderstood. MicroRNAs (miRNAs) have been implicated in the regulation of geneexpression of various biological processes in a post-transcriptional manner underphysiological and pathological conditions including host responses to viral infections.Increasing evidence indicates that microRNAs have important functions in viralreplication and may be used by host cells to inhibit or promote viral infections.In previous mices were used in vivo or in vitro studies for PHEV. CNS is theultimate infection target of PHEV and the virus cause the damage to cerebral cortex.The cerebral cortex is rich in miRNA. Changes in the expression of many genes wereat the same time with changes in the expression of miRNA during the period ofPHEV-infected the cerebral cortex.To study the changes of mRNA or microRNA expression profile in mice cerebralcortex infected with PHEV, a mice infection model with PHEV was established. Allmice so examined were killed at3or5dpi, just as PHEV antigen was first detected inmice cerebral cortex or the typical clinical signs of PHEV became aggressive ininfected animals. To gain insight into the interaction between virus and host cells, Theglobal gene expression changes in the cerebral cortex of PHEV-or mock-infectedmice in3dpi and5dpi were investigated using DNA microarray analysis andmicroRNA microarray analysis and quantitative real-time PCR assay. The results ofmicroarray analysis showed that365genes on day3post infection (upregulated,204;downregulated,161) or781genes on day5post infection (upregulated,508; downregulated,273) and24miRNA (upregulated,17;downregulated,7) or14miRNA(up-regulate,13; downregulated;1) in cerebral cortex were differentially expressed inresponse to PHEV infection.By bioinformatics analysis, we found that upregulated genes were related to theimmune response and host defense, such as antigen processing and presentation、Jak-STAT signaling pathway、 RIG-I-like receptor signaling pathway、 Toll-likereceptor signaling and apoptosis-related proteases etc, however obviouslydown-regulated genes are manly involved in nervous system development, such assynaptic transmission, neuron projection development, transmission of nerve impulseand negative regulation of glial cell differentiation, etc. Furthermore, bioinformaticanalyses of the differentially expressed mRNAs and miRNAs were carried out tointegrate miRNA and mRNA expression and revealed that alterations in mRNAfollowing PHEV infected were negatively correlated with miRNA expression. Thesefindings significantly advance our understanding of the regulatory mechanismsunderlying the pathophysiology of PHEV-induced brain injury specifically withregard to miRNAs. The results showed that the function of different microRNArelated different genes were mainly involved in regulating immune response, axonguidance and cell apoptosis etc. In a word, the differential expression of these genesand microRNA suggests a strong antiviral response of host but may also contribute tothe pathogenesis of PHEV resulting in encephalomyelitis.Apoptosis is a tightly controlled multistep mechanism of cell death that ischaracterized by chromatin condensation, plasma-membrane blebbing, cell shrinkage,and DNA fragmentation into membrane-enclosed vesicles or apoptotic bodies. Thereis evidence suggesting that apoptosis is closely related to nerve injury. In our study wediscovered that PHEV infection can induce mouse cerebral cortex injury andapoptosis-related proteases were up-regulated. However, the mechanisms inducingdeath in PHEV-infected cells remains largely unknown. Our results demonstrate thatPHEV can induce apoptosis of PK-15cells or N2a cells by the observation ofapoptosis such as PHEV-induced cytopathic effect (CPE)、 PHEV-inducedmorphological changes in the nuclei and PHEV-induced DNA fragmentation etc.Furthermore, our results suggested that PHEV can activate FasL-mediated deathreceptor pathway and mitochondrial-mediated pathway to induce apoptosis in N2acells. Lastly, our results suggested that PHEV-indued cell apoptosis require virus replication.By contrast, it’s not necessary.On the other hand, our result showed that the amount of mmu-let-7b expressionin PHEV-induced apoptosis of N2a cells was down-regulation. According tobioinformatics database and research report, procaspase3gene is a target gene of let-7family.In view of this, mmu-let-7b mimics and inhibitor were synthesized andtransfected into N2a cells respectively, and then inoculated with PHEV. To study ofinterenation of mmu-let-7b, apoptosis (procaspase3) and virus,the methods ofdetecting the activity of caspase3, TCID50and so on were applied. Results: Downregulated of mmu-let-7b during PHEV replication and propagation result to theprocaspase3protected and which indirectly promoted expression of activation ofcaspase3and PHEV-induced neuronal apoptosis and the release of PHEV.In conclusion, the first application of gene chip technology and bioinformaticsmethods by us, to screen for different miRNAs and genes after PHEV infection incerebral cortex of mice, and to establish a regulatory network of different miRNAsand genes. From miRNA and mRNA angle, we investigate that the molecularmechanism of cerebral cortex injury induced by PHEV and provide researchfoundation for the following test. In addition, Our study showed that PHEV caninduce cell apoptosis and preliminary study of signal pathway and regulationmechanism of mmu-let-7b in PHEV-induced cell apoptosis. Our result elucidate thevaried transcriptional responses of PHEV virus-infected in cerebral cortex and thesetranscripts provide a better understanding of the pathogenesis of PHEV at atranscriptomic level, particularly the molecular events that underpin host defensemechanism against PHEV infection.