The Effect Of MiR-21a-5p On PHEV Replication And Its Mechanism

Porcine hemagglutinating encephalomyelitis virus(PHEV) has the typical characteristics of neurotropic coronavirus, belonging to the Betacoronavirus genus within the Coronaviridae family. PHEV infects mainly piglets under the age of 3 weeks and causes vomiting, diarrhea, and obvious neurological symptoms.The mortality rate ranges from 20 to 100%. It will cause huge economic losses to the pig industry when the outbreak of the disease, because, currently, no effective preventative measures or cures exists for this disease. PHEV is a highly neurovirulent virus that spreads to the central nervous system via peripheral nerves but the mechanism induced by nerve injury is unclear. It has great scientific significance to study the pathogenesis of PHEV from the point of view of virus infection and host protein interaction. Micro RNAs(mi RNAs) constitute a class of 22 nucleotides short non-coding RNAs that play an important role in the course of many biological and the development of disease. Mi RNA plays a regulatory role in the course of Virus replication, proliferating and release process by regulating the expression of target genes, such as HSV-1, influenza virus, hepatitis C virus. The host mi RN A expression profiles appear great changes in PHEV infected brain tissue by using of gene chip, suggesting that these differentially expressed in the host mi RN As may play an important role in the process of the virus infection. This provides a new way to study the pathogenesis of PHEV.In this study, we chose mi R-21a-5p as the research object. Mi R-21 expressions during PHEV infection process were determined by quantitative real-time PCR. The results showed that the expression of mi R-21a-5p was significantly up-regulated compared with the control group, suggesting that it might be involved in the regulation of PHEV infection. To study the effects of mi R-21 on virus replication, N2 a cells were transfected with mi R-21a-5p mimics or inhibitor respectively, then treated with PHEV. The content of PHEV genomic RN A was detected by quantitative real-time PCR. The results confirmed that overexpression or down-regulated mi R-21a-5p in N2 a cells could decrease or increase the copy of PHEV proliferation. Thus, in the process of PHEV infection, mi R-21a-5p promoted the viral genome replication in cell level.In order to further clarify the target of mi R-21a-5p in replication of PHEV. Potential target genes of mi R-21a-5p were predicted by Target Scan、Microcosm Microcosm、Pic Tar databases and the function of the target gene s were analyzed. Scaffold protein Caskin1 which participates in the regulation of synaptic transmission, axon guidance and the stability of the cytoskeleton in the maintenance of neuronal cells may play a role in nerve function injury caused by PHEV. To test whether Caskin1 is a direct target of mi R-21a-5p in the course of PHEV infection, we prepared an Dual- luciferase reporter expression system using the 3’-UTR of Caskin1 containing the putative mi R-21 binding site and using 3’-UTR of Caskin1 containing a mutant mi R-21 binding side to verify; the m RNA and protein expression of Caskin1 in N2 a cells were tested after transfecting with mi R-21a-5p mimics or inhibitor. Taken together, these results indicate that mi R-21a-5p negatively regulates Caskin1 expression in the course of PHEV infection in host cell by directly targeting the 3’-UTR of the Caskin1 gene. To explore whether mi R-21a-5p promote PHEV genome replication through targeting Caskin1, N2 a cells were transfected with Caskin1 si RNA, then treatment with PHEV. The expression of viral genome quantity increases obviously, indicating that mi R-21a-5p can promote PHEV genome replication in host cel s by regulating the expression of Caskin1.To determine the effect of mi R-21a-5p on mice in the pathogenic process of PHEV, the mice were treated with mi R-21a-5p antagomir, mi R-21a-5p antagomir NC, PBS by intracerebral injectio, normal mice as control mice. We observed the changes of body and body weight in mice after Infecting PHEV, and detected the content of PHEV genomic RN A and Caskin1 expression in mouse brain when 5 days ago. The results showed that mi R-21a-5p antagomir can effectively delay the onset time of PHEV infected mice, and inhibit the replication of PHEV in the brain tissue of mice. Meanwhile, the expression of Caskin1 in mice brain tissue Caskin1 was increased, suggesting that Caskin1 is a target gene of mi R-21a-5p in vivo.Taken together, these results indicated that decreasing the concentration of mi R-21a-5p in the brain tissue of mice can reduce the PHEV infection of pathogenic process, and mi R-21a-5p can promote PHEV genome replication in the brain tissue of mice by regulating the expression of Caskin1.