Differential Expression Of MiR-375/499and Validation Of Predicted Target Genes In The Testicular Tissue Of Swine At Different Developmental Stages

MicroRNAs (miRNAs) are classes of endogenous small non-coding sequenceswhich widely existed in tissues and organs. And miRNAs play important role in manyprocesses by their target gene degradation or hindered. Studies have shown thatmiRNAs in testis regulated the development of the sminiferous tubules and spermmaturation. Also, miR-375and miR-499were identified difference expressionbetween testicular tissue of1-month-old and6-month-old. Then, in this study, wedetected the expression of miR-375and miR-499in testis at different developmentalstages and validated their target genes.HE staining was observed in several stages testes of Junmu NO.1swine. Thediameter of seminiferous tube gradually increased and the number of tube cavityreduced with the increasing of age. And the supporting cells and spermatocytesgradually moved to the basement membrane of seminiferous tube cavity.At the same time, the expressions of miR-375/499and their target genesDIAPH2, YAP1, YBX1, ITPKB, NR5A2and CYLC1, DMRT1, QKI, XRN2, ZNF313were detected by real time quantitative fluorescence in testis of swine. The resultsshown that the expression of miR-375was low in1-day of testis;1-4month old oftestis; while in4-7month showed a significantly increasing. The expression ofmiR-499was upregulated in1-day,1-2month, and then showed a downward in2-7month of age. The expressions of target genes of miR-375and miR-499weredifferentially expressed in testicular tissue at different developmental stages.SPSS13.0analysised shown that miR-375and target gene DIAPH2was existedsignificantly negative correlation. miR-499and target gene QKI, CYLC1existedsignificantly negative correlation.Then the protein expression of QKI gene was detected in testis at differentmonths. Higher expression in1-day and5-7month was founded by immunoblotting. Therefore, the luciferase reporter gene system was used to verify correlationbetween miR-499and QKI gene. Activity of luciferase was significantly lower inmiR-499co-transfected with pmiR-RB-REPORT-QKI-WT group than the miR-499co-transfected with pmiR-RB-REPORT-QKI-mut/si groups indicating that targetsequence of miR-499was existed in3’UTR of QKI gene.Furthermore, the expressions of miR-499and QKI were detected in testicularcells which transfected miR-499, miR-499negative control and untreated. The resultsshowed that diameter of convoluted seminiferous tubule growth with the increasedage. Significantly different expressions of miR-499and its target genes were foundedin swine testicular tissue at different developmental stages (P<0.05), and alsooverexpressing miR-499analysis suggested that miR-499was negatively correlated tothe expression of QKI (P<0.05). In conclusion, QKI is a target gene of miR-499. Andcombined with development of testicular tissue, we speculated that miR-375/499andtheir respective target genes differentially expressed may be linked to thedevelopment of testicular tissue and spermatogenesis.