MiR-125b Suppress PRRSV Through Negatively Regulates NF-κB Pathway

Porcine reproductive and respiratory syndrome also known as "blue ear" disease has brought great harm and heavy economic losses since first reported in 1987 with the United States and has known in the important pig breeding Conutry. Porcine reproductive and respiratory syndrome virus is main pathogen of this disease.The virus contain nine openning coding areas and the nocoding areas on the end of the genes, natural infection mainly in pig alveolar macrophage, what's more, artifical infection aslo in few of cells.such as PAM, Marc-145, CL2621 et al. In recent years, the studies found that miRNA widely participated in the progress of virus infected cells, but the regulation to PRRSV proliferation by miRNA still had no report related. Ten miRNAs have been selected through refer to a large number of documents, involved in the widely regulation of immunity, the more concervaty and direct control the viral repliction.This study detected whether exist the inner miRNAs in cells which played the regulator role in the progress of PRRSV replications. This study is more important for the further detection of the molecular biology mechanism of PRRSV copy and lay the foundation for related biological principle pathogenic. Take all above into account,this study was to investigate the following aspects.1.miRNA selected inhibited the proliferation of PRRSV.We found mang miRNAs which affected the determination of PRRSV proliferation and its role in Marc-145 by involving large number of documents, and transfected the miRNAs which were synthesised by JIMA company into Marc-145 and PAM. we designed the experiments about the miRNA expression in porcine alveolar macrophages (PAM) because of the clinical value. Through the bite spot experiment and RT-PCR, we found that the miR-125b has the activity in inhibiting the proliferation of PRRSV in Marc-145 and PAM. At the same time, indirect immunofluorescence tested N proteins and western-blot tested the expression of PRRSV Nsp2. All experiments had do doses studies and showed that miR-125b has the activity about antiviral. 2.Selecte target genes of miR-125b which have been locused on and the relationship among miR-125b,кB-RAS2 and NF-кB.Predicted by main miRNA target genes detection software and constructed the fluorescent element enzyme report plasmid, which showed that miR-125b does not act on the PRRSV 3'UTR, while do not act on all the coding region of genes. In host aspect, we predicted most of the action sites such asкB-RAS2, TNFa, ISATA3 and so on. In This study, we considered the NF-кB signaling pathway and co-transfected miR-125b and NF-кB-1uc, which found that the miR-125b inhibit the NF-кB transcription.3.The relationship between the proliferation of PRRSV and NF-кB expression.Experiments mainly adopted NF-кB transcription inhibitor BAY11-7082 to treat the cells. PRRSV plaque experiments detected the influence of the treatment, of coures we transient transfected miR-125b and p65 in Mrac-145 cell lines. We were pleased to found that p65 can reverse the inhibitory of virus proliferation by miRNA transfected. And the study showed that the NF-кB transcription was more profit for copies of PRRSV.