miRNAs are involved in various biological processes, such as host-virus interactions and antiviral immunity. In this study, we investigated the role of miR-29 on porcine reproductive and respiratory syndrome virus (PRRSV) replication and its target genes. At first, miR-29a/b-1/c expression was detected when porcine alveolar macrophages (PAMs) were infected with PRRSV at different infective doses by real time-quantitative polymerase chain reaction (RT-qPCR). The result showed that miR-29a/b-1 expression significantly increased after 6 h (p < 0.01), with the peak around 24 h, miR-29c expression in each period of PRRSV infection was very low. Then, pre-miR-29a/b-1 lentiviral vectors were constructed. Absolute RT-qPCR analysis showed that PAMs transfected with pre-miR-29a/b-1 lentiviral vectors significantly promoted PRRSV replication in PAM within 24 h (p < 0.01). The expression of the target genes (AKT3, TP53INP1, and RPS6KB1) of miR-29a significantly reduced (p < 0.01). Western blot analysis showed that AKT3 and TP53INP1 are reduced at miR-29a overexpression. To further validate the interaction between miR-29a and its target gene sites, the luciferase assay results demonstrated that miR-29a interacted with AKT3 3'UTR 1676 and 1261 sites, leading the inhibition of luciferase expression. Our findings support that miR-29a could promote PRRSV replication during early stage of virus infection in vitro and AKT3 could be the target gene of miR-29a.

microRNAs (miRNAs) represent a newly identified class of nonprotein-coding *22 nt small RNA that plays important roles in multiple biological processes by degrading targeted mRNA or repressing mRNA translation. This study observed the morphology of swine testicular tissue at different developmental stages (including 1-day old, 1-7 month old) by Hematoxylin-eosin staining. We also examined the expression of miR-499 and its target genes (CYLC1, DMRT1, QKI, XRN2, ZNF313) in samples of tissue slices using quantitative reversetranscription polymerase chain reaction, which showed that miR-499 had a significant negative correlation with QKI gene. Then, the proteins of QKI gene expression were determined by western blot, which were consistent with results of quantitative polymerase chain reaction (qPCR) detection. Therefore, the luciferase reporter gene system was used to verify correlation between miR-499 and QKI gene. Activity of luciferase was significantly lower in miR-499 co-transfected with pmiR-RB-REPORT-QKI-WT group than the miR-499 co-transfected with pmiR-RB-REPORT-QKI-mut/si groups, indicating that target sequence of miR-499 existed in 3¢UTR of QKI gene. Furthermore, the expressions of miR-499 and QKI were detected in testicular cells that were transfected with miR-499, miR-499 negative control and untreated. The results showed that the diameter of convoluted seminiferous tubule growth increased with age. Significantly different expressions of miR-499 and its target genes were found in swine testicular tissue at different developmental stages ( p < 0.05), overexpressing miR-499 analysis, suggesting that miR-499 was negatively correlated to the expression of QKI ( p < 0.05). In conclusion, QKI is a target gene of miR-499.

To verified the target genes of miR-34c, bioinformatics software was used to predict the targets of miR-34c. Three possible target genes of miR-34c related to spermatogenesis and male reproductive development: zinc finger protein 148 (ZNF148), kruppel-like factor 4 (KLF4), and platelet-derived growth factor receptor alpha (PDGFRA) were predicted. Then, the expression of miR-34c and its target genes were detected in swine testicular tissue at different developmental stages by quantitative polymerase chain reaction. The results suggested that the expression of PDGFRA has the highest negative correlation with miR-34c. Then immunohistochemical staining was done to observe the morphology of swine testicular tissue at 2-days and 3, 4, 5-months of age, which indicated that PDGFRA was mainly expressed in the support cells near the basement membrane during the early development stages of testicular tissue, but that the expression of PDGFRA was gradually reduced in later stages. Therefore, western blot analyzed that the highest expression of PDGFRA was generated in 2-days old testicular tissues and the expression levels reduced at 3 and 4-months old, which correlated with the results of immunohistochemical staining. In conclusion, PDGFRA is a target gene of miR-34c.