Selection Of Swine Micrornas Inhibiting 3'UTR Of Porcine Reproductive And Respiratory Syndrome Virus In Suvec In Vitro

MicroRNAs (miRNAs), which are widely observed in all kinds of organism, are single-stranded RNAs of 22-nucleotides in length that play a critical role in regulating gene expression in multicellular organisms. MiRNA guides gene silencing by base pairing mainly with the 3'-untranslated region (3'UTR) of the target mRNAs, and leads to translational repression and/or mRNA cleavage. In recent years, many studies found out the important roles of miRNA during viral infection. Virally encoded miRNAs can cis-regulate or trans-regulate gene expression in host cells, and the cellular miRNAs can also be positive or negative regulation of viral replication and its gene expression. There were no reports relating to miRNA regulation during porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, swine miRNAs have undergone a preliminary selection and verification during PRRSV infection for investigating the role of miRNA.1. Target prediction of swine miRNA on PRRSV 3'UTR. We obtained 76 swine miRNA sequences from miRBase (miRBase 15.0, 2009). Potential miRNAs targeting PRRSV 3'UTR were predicted by bioinformatics, and the target prediction softwares included miRanda, RNAhybrid and RNA22 microRNA target detection. Three potential miRNAs (ssc-miR-323, ssc-miR-105-1, ssc-miR-105-2) were obtained, from which ssc-miR-323 and ssc-miR-105-1 were selected for artificial synthesis.2. Construction of the recombinant vector, psiCHECK-UTR. PRRSV (NVDC-JXA1) was inoculated into cultured Marc-145 cell lines for proliferation. JXA1 3'UTR sequence was obtained from GenBank (Accession No.EF112445). We designed the primer, extracted viral RNA and amplified 3'UTR sequence, and inserted the PRRSV 3'UTR into psiCHECKTM-2 plasmid. Sequencing of dual-luciferase recombinant vector, psiCHECK-UTR, verified that the recombinant vector was successfully constructed.3. Co-transfection of miRNA and psiCHECK-UTR into swine umbilical vein endothelial cells (SUVEC) and verification of the miRNA function. The dual-luciferase vector was co-transfected accompanying with synthesized miRNAs or inhibitors into SUVEC. The Dual-Glo Luciferase activity was detected by luminometer and the relative activity was analyzed by data processing. This result showed that relative activity of dual-luciferase did not decline after ssc-miR-323 was co-transfected accompanying with psiCHECK-UTR into SUVEC, and ssc-miR-323 inhibitor did not recovery the relative activity. However, the ssc-miR-105-1 reduced the relative activity of dual-luciferase and ssc-miR-105-1 inhibitor recoveried the relative activity. All these results indicated that ssc-miR-323 has no effect on PRRSV 3'UTR expression, whereas the ssc-miR-105-1 has a little influence on inhibiting the expression of PRRSV 3'UTR.