Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (22): 4389-4397.doi: 10.3864/j.issn.0578-1752.2017.22.015

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Regulation of the BCKDHB Gene Expression by miR-433-3p in Ovine Preadipocytes

YAN XiaoRu1, SHI Tao1, PAN YangYang1, JING JiongJie1, CHENG LiFen2, CAO NingXian2, QIAO LiYing1, LIU WenZhong1   

  1. 1College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi2Division of Animal and Poultry Breeding, Department of Agriculture of Shanxi Province, Taiyuan 030001
  • Received:2017-06-20 Online:2017-11-16 Published:2017-11-16

Abstract: 【Objective】 This study aims to reveal the target relationship between miR-433-3p and BCKDHB by theoretical prediction and experimental verification approaches. 【Method】The binding site between miR-433-3p and BCKDHB was predicted by three bioinformatics softwares including TargetScan、miRanda and DIANA-microT based on the sequence of BCKDHB. In order to get the target fragment the primersof BCKDHB 3′-UTR were designed for PCR amplification. Double digestion with Xho I and Xba I was performed with vector Pmir-GLO and target fragments, which were then ligated with T4 enzyme to obtain the recombinant dual-luciferase expression plasmid. Two vectors of mimics and NC were commercially available. One experimental (overexpressing miR-433-3p) and two control groups (negative and blank) were set. By using LipofectamineTM3000 reagent the dual-luciferase vector was co-transfected with the commercially obtained vectors into HEK-293T cells of overexpressing-miR-433-3p and negative groups, respectively. And the cells of blank group were cultured normally. Subsequently, their relative luciferase activities were detected, with renilla luciferase activity as the control. Ovine preadipocytes were cultured in vitro to study the interacting relationship of miR-433-3p and BCKDHB in the differentiated preadipocytes. MiR-433-3p overexpression was adopted to study its regulating function to BCKDHB by detecting RNA quantity of miR-433-3p and BCKDHB as well as protein quantity of BCKDHB before and after miR-433-3p overexpression in ovine preadipocyte. RT-qPCR was carried out to detect the temporal expression dynamics of BCKDHB and miR-433-3p in the differentiation process of ovine preadipocytes . In order to increase the reliability of the results, we also carried out photo collection and oil red O staining at different stages during the differentiation of ovine preadipocytes.【Result】 The results showed that miR-433-3p had a theoretical binding site at the 8~28 bases of BCKDHB 3′-UTR. We found miR-433-3p overexpression significantly (P<0.01) decreased luciferase activity of the recombinant double fluorescent plasmid by comparing the relative fluorescence activity of the overexpression, the negative and the blank groups, indicating that miR-433-3p could bind to the 3′-UTR, which verified the correctness of our prediction. Overexpressed miR-433-3p inhibited the expressions of BCKDHB at both mRNA (P<0.01) and protein (P<0.05) levels in ovine preadipocyte. The expression of miR-433-3p correlated negatively with that of BCKDHB in the process of ovine preadipocyte differentiation. Cellular photos and the oil red O analysis showed lipid droplets accumulated in the differentiation of ovine preadipocytes.【Conclusion】 These results indicated that miR-433-3p negatively regulated the expression of BCKDHB and its coding protein by binding to the 3′-UTR, which provided a scientific basis for further research on the molecular mechanism of BCKDHB in regulating fat metabolism in sheep.

Key words: miR-433-3p, BCKDHB, ovine, preadipocytes, cell differentiation

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