| Deposition of intramuscular fat(IMF)is distributed like white marbling,it is one of the most important traits for the evaluation of beef carcass quality grade.The richer marble pattern with higher IMF in beef,so the quality of beef is superior.The deposition of IMF is regulated by genetics,genes,and epigenetics,among these microRNA(miRNA)is an endogenous non-coding RNA composed of 19-22 nucleotides that can play a role in the post-transcriptional regulation of mammalian preadipocyte differentiation.Previously,bta-miR-130 a regulates milk fat biosynthesis by targeting PPARG.However,the role of miR-130 in the regulation of bovine adipocyte differentiation remains unknown.Based on the above background,this research is going to make sure the role and the molecular mechanism of bta-miR-130a/b in the differentiation of Qinchuan bovine preadipocytes.In this study,Qinchuan beef cattle preadipocytes were first isolated,then we investigated the function of bta-miR-130a/b during preadipocytes differentiation by transfecting bta-miR-130a/b mimics or inhibitor,respectively.Mechanically,we used bioinformatic analysis,dual luciferase report assays and in vitro rescue experiments to predict and verify the target genes of bta-miR-130a/b on differentiation of preadipocytes.The main results of this study are shown as follows:1.Bta-miR-130a/b inhibits preadipocytes adipogenesis and decreases triacylglycerol levels.Overexpression of bta-miR-130a/b led to significantly decreased cellular triacylglycerol(TAG)levels during adipogenesis process as well as reduced lipid droplet formation.In contrast,the inhibition of bta-miR-130a/b resulted in larger lipid droplets and TAG accumulation.2.Bta-miR-130a/b regulates differentiation-related genes in adipocytes.In addition,overexpression of bta-miR-130a/b inhibited the expression of adipocyte differentiation-related genes,including C/EBP?,C/EBP?,FABP4,LPIN1,and LPL.While bta-miR-130a/b was decreased,the expression of C/EBP?,C/EBP?,FABP4,LPIN1 and LPL were increased.3.Bta-miR-130a/b directly targets PPARG for negative regulatory.When mRNA and protein levels were detected,the expression of PPARG was significantly decreased when bta-miR-130a/b was increased,whereas the expression of PPARG was increased when bta-miR-130a/b was decreased;Dual luciferase reporter vector activity studies revealed that bta-miR-130a/b mimics significantly inhibited PPARG wild-type luciferase reporter vector activity;In vitro rescue assays showed the corresponding quantitative visualization analysis further indicated the preadipocytes with co-transfection of bta-miR-130a/b inhibitor and si-PPARG had less lipid droplets compared with the preadipocytes that transfected with bta-miR-130a/b inhibitor only.Therefore,bta-miR-130a/b can affect the differentiation of adipocytes by directly targeting PPARG for negative regulation.4.Bta-miR-130a/b directly targets CYP2U1 for positive regulatory.When mRNA and protein levels were detected,the expression of CYP2U1 was significantly increased when bta-miR-130a/b was increased,whereas the expression of CYP2U1 was decreased when bta-miR-130a/b was decreased;Dual luciferase reporter vector activity studies revealed that bta-miR-130a/b mimics significantly inhibited CYP2U1 wild-type luciferase reporter vector activity;In vitro rescue assays showed the corresponding quantitative visualization analysis further indicated the preadipocytes that co-transfected with both bta-miR-130a/b in-hibitor and si-CYP2U1 had more lipid droplets than the preadipocytes only transfected with bta-miR-130a/b inhibitor.Therefore,bta-miR-130a/b can affect the differentiation of adipocytes by directly targeting CYP2U1 for positive regulation.Based on pervious experiments,our study shows that bta-miR-130a/b play important role in the regulation of adipocyte differentiation by targeting the 3'UTR of PPARG and CYP2U1.Our result provides a reference for illustrating how noncoding RNAs affect beef quality traits in beef cattle. |
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