Prediction And Analysis Of MiRNAs And Their Target Genes Related To Adipocyte Differentiation Of Yak

Yak (Bos grunniens) is mainly distributed in the Qing-Tibet Plateau and its adjacent areas. The traditional feeding system and unique environment were proposed to contribute to the various aspects of meat quality, and lead to the adipose tissue of yaks grow rapidly owing to plenty of pasture in summer, while in cold winter with a shortage of pasturage, the adipose tissue of yaks suffer severe weight loss. Adipocyte differentiation has an important effect on the growth of adipose, which is regulated by many factors. Some scientists found that miRNAs have been proved regulate the process. The developed mechanism is not yet understood in yak. In order to we unravel genetic mechanism of adipocyte differentiation in yak. In this study, we predicted miRNAs, which might take part in adipocyte differentiation, and then we analyzed candidate miRNAs' expression level in different tissues. Using TargetScan and STRING predicted their target genes and analyzed functions. The dual-luciferase reporter system was used to identify target genes. The results are as follows:1. Screening miRNAs related adipose differentiation of yakFirst we predicted cattle miRNAs about adipose differentiation. Using Blast got yak miRNAs, and Mfold predicted five pre-miRNAs. We got bta-miR-2340, bta-miR-1260b, bta-miR-22-5p, bta-miR-2328-3p, bta-miR-1777b and bta-miR-1388-5p maybe took part in adipose differentiation of yak.2. Expression analysis of miRNAs between differential organizations.We chosed miRNAs identified by computational method for expression analysis by RT-PCR. The results show that bta-miR-2340, bta-miR-1777b and bta-miR-1260b expressed in fat higher than the other tissues, bta-miR-2328-3p, bta-miR-1388-5p and bta-miR-22-5p were the opposite. However adipose, liver and pancreatic tissues were main organization of lipid metabolism. So we predicted bta-miR-2340, bta-miR-1777b and bta-miR-1260b maybe regulate adipocyte differentiation.3. Predicted targeted genes of miRNAs and analyzed functionsPredicting above candidate miRNAs targeted genes by TargetScan. Bta-miR-2340 and bta-miR-1777b got 131 and 49 respectively, and bta-miR-1260b got no mRNA. STRING was used function analyze for mRNAs. Bta-miR-2340 targeted mRNAs include BAD, CHRM1, IRAK1, SOCS3 and FOXO4, Who major participant in adipocyte differentiation.4. qRT-PCR of bta-miR-2340 targeted genesDetecting differential expression of BAD, CHRM1, IRAK1, SOCS3 and FOXO4 were in tissues. We know that BAD and IRAK1 were relatively highly expressed in different tissues. BAD expressed in heart higher than others. Compared with expressed in fat tissue, CHRM1 was highly in kidney. The expression level of IRAK1 in heart and spleen were highly, and others tissues were all lowly. SOCS3 expressed highly in lung and fat. FOXO4 in heart and dorsal expressed higher than others. But BAD and IRAK1 were relatively lowly in all tissues.5. Identifying of bta-miR-2340 target geneWe used dual-luciferase reporter gene system to identifying target genes. We respective building BAD, CHRM1, IRAK1, SOCS3 and FOXO4 genes were selected for dual-luciferase reporter gene system, and transfected into Hela cell under bta-miR-2340 mimics and lipo 2000. The results show that bta-miR-2340 mimics decrease BAD mutant type reporter, and no effect to CHRM1, IRAK1, SOCS3 and FOXO4 mutant type reporters. But bta-miR-2340 mimics also didn't influence CHRM1 wild group. The luciferase activity of SOCS3 wild type reporter was decreased about 20%. So we consider none was bta-miR-2340 target gene.