Differential Expression of miR-499 and Validation of Predicted Target Genes in the Testicular Tissue of Swine at Different Developmental Stages

microRNAs (miRNAs) represent a newly identified class of nonprotein-coding approximate to 22nt 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 reverse-transcription 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 3UTR 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.