The Effect Of MiR-29on PRRSV Replication In PAM And The Certification Of Its Targets

Porcine reproductive and respiratory syndrome（PRRS）is one of the mostimportant infections in the swine industry at present, which have the characteristicsof highly pathogenic and high fatality rate. PRRS causes significant reproductivefailure and respiratory disease in infected swinery, huge economic losses every year.The pathogene of PRRS is PRRSV. As a RNA virus, the genome of PRRSV have ahigh rate of genetic variation, that can escape from the immune mechanism of host.Given the important role in the interaction of host-virus, our study chose a differentexpressed miRNA family--miR-29as the object to investigate the effect of miR-29on PRRSV replication. This study will for the exploration of infection mechanism ofPRRSV and anti-PRRSV infection mechanism of host. This study include thefollowingaspects:A.The effect of PRRSV replication on miR-29expression in PAM.We infected PAM with different doeses PRRSV,0.8MOI,1.0MOI and1.2MOI,and determined the expression of miR-29at0hpi、6hpi、12hpi、24hpi、36hpi and48hpi. The results showed the expression level of miR-29a began to increase afterPRRSV infection at6h time point, but the difference was not significant. Thedifferences were very significant at the time points of12h,24h,36h and48h. Theexpression trends of miR-29b-1and miR-29a were almost identical. While themiR-29c was not expressed or had a low expression level in PAM and could not beidentified by qPCR.B.The effect of miR-29a/b on the PRRSV replication.PAM cannot be passaged and difficult to transfect small moleculeswre asnon-breeding cells. In this study,the pre-miR-29recombinant lentivirus particles wasconstructed. The recombinant lentivirus was used for PAM infection. The PRRSVinfection was done after the expression of MiR-29was stable. The PRRSVreplication was detection by absolute quantitative. The results showed that MiR-29a could significantly promote the PRRSV replication in PAM. The role of miR-29aand miR-29b-1over-expression were similar. Meanwhile, the overexpression ofmiR-29a could inhibit AKT3, TP53INP1and RPS6KB1expression levels.C.The certification of targets of miR-29.The fragment of3’UTRof AKT3, RPS6KB1containing miR-29a seed sequencebinding site were inserted in psiCHECK-2dual luciferase reporter gene vector. The3’UTR of targets included two action sites. While the reporter gene vectors wereconstructed by the mutation of miR-29a seed sequence binding site.The relativelevels of fluorescence were detected after co-transfection of Marc-145cells withpre-miR-29a recombinant lentivirus, blank lentivirus and psiCHECK-2WT/MTplasmid. The results showed that miR-29a might act directly on the3’UTR of AKT3gene to inhibit their expression. Meanwhile, miR-29a does not act on the PRRSVgenome and3’UTR of RPS6KB1directly.