Differential Expression And Function Of MiR-122 And MiR-449a In Red Steppe Testis Development

To explore the association of miRNA with testicular tissue. The aim of the study is to investigate the differential expression of miR-122,miR-185,miR-146 b,miR-140,mi R-449 a,miR-204,miR-375 and miR-221 of red steppes testicular tissue and other tissues among different developmental stages by Real-time PCR, to screen specific expressed or tendentious expressed miRNA in testicular tissue. Furthermore, target genes of these miRNAs were predicted by forecast software to construct dual luciferase reported vector, the results of dual luciferase reporter assay in fat cells of red steppe are shown as followings:The relative quantitative expression of miR-122,mi R-185, miR-146 b,miR-140, mi R-449 a, miR-204, miR-375 and miR-221 had great differences in different tissues in red steppe through analyzing relative expression quantity. Among them, miR-122 and miR-449 a relatively highly expressed in testicular tissue,There were some characteristics in the expression of miR-122 and miR-449 a in testicular tissue, besides, the expression level of miR-122 between testicular tissue and other tissues had extremely significant difference.The expression level of the 8 miRNA were on varied degree in testicular tissue in different developmental stages.miR-221 and miR-204 relatively highly expressed in the calves,the results indicated that mi R-140 and miR-185 highly expressed in the red steppe’ testicular tissue of 12 months ages, miR-449 a, miR-375, mi R-122 and miR-146 b and relatively highly expressed in the red steppe’ testicular tissue of 24 months ages, and the Real-time PCR results of the above nine miRNA were consistent with those of early birth and 24 months of solexa bulls to high-throughput sequencing results. Also, the up-regulation of miR-122,miR-146 b,miR-449 and miR-375 and the down-regulation of miR-185,miR-140, miR-204 and miR-221 were observed in testicular tissue in red steppes.The promising target genes of miR-122 may be EPO,MAF1,PDK4,DUSP4,FUT8, PRKRA and mi R-449 a by software, FKBP1 B may be the target genes of miR-449 a. The 3’UTR were amplified by PCR and then the dual luciferase reported vector were constructed by inserting 3’UTR into PGL3-control vector, and transferred into bovine fat cells with pre-mir. The results shows that DUSP4 and FKBP1 B may be the target gene of miR-122 and miR-449 a, respectively, by measuring the dual luciferase activity with enzymatic analyzer.In conclusion, This study analyzed the expression differences of miRNAs in different growth period in testicular tissue and other tissues in red steppe and forecasted the target genes of miR-122 and mi R-449 a with high expression in testicular tissue that was confirmed in vitro.The results play an important role on the study of the development of reproductive organs and the regulation mechanism of reproductive activity of miRNA in beef cattle.