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Asian-Australas J Anim Sci > Accepted Articles
DOI: https://doi.org/10.5713/ajas.19.1000    [Accepted] Published online April 12, 2020.
Regulation of chicken Vanin1 gene expression by PPARα and MiRNA-181a-5p
Zhongliang Wang1,2  , Jianfeng Yu1  , Nan Hua1  , Jie Li1,2  , Lu Xu1  , Wen Yao2  , Zhiliang Gu1,* 
1School of Biology and Food Engineering, Changshu Institute of Technology, Changshu, 215500, Jiangsu, China
2College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, Jiangsu, China
Correspondence:  Zhiliang Gu, Tel: +86-512-52251568, Fax: +86-512-52251568, Email: zhilianggu88@hotmail.com
Received: 31 December 2019   • Revised: 24 February 2020   • Accepted: 30 March 2020
Abstract
Objective
Vanin1 (VNN1) is a pantetheinase that can catalyze the hydrolysis of pantetheine to produce pantothenic acid and cysteamine. Our previous studies showed that VNN1 is specifically expressed in chicken liver. In this study, we aimed to investigate the roles of peroxisome proliferators activated receptor α (PPARα) and miRNA-181a-5p in regulating VNN1 gene expression in chicken liver.
Methods
5'-RACE was performed to identify the transcription start site of chicken VNN1. JASPAR and TFSEARCH were used to analyze the potential transcription factor binding sites in the promoter region of chicken VNN1 and miRanda was used to search miRNA binding sites in 3' untranslated region (3'UTR) of chicken VNN1- We used a knock-down strategy to manipulate PPARα (or miRNA-181a-5p) expression levels in vitro to further investigate its effect on VNN1 gene transcription. Luciferase reporter assays were used to explore the specific regions of VNN1 targeted by PPARα and miRNA-181a-5p.
Results
Sequence analysis of the VNN1 promoter region revealed several transcription factor-binding sites, including hepatocyte nuclear factor 1α(HNF1α), PPARɑ and CCAAT/enhancer binding proteinɑ (CEBPɑ). GW7647 (a specific agonist of PPARα) increased the expression level of VNN1 mRNA in chicken primary hepatocytes, whereas knockdown of PPARα with siRNA increased VNN1 mRNA expression. Moreover, the predicted PPARα-binding site was confirmed to be necessary for PPARα regulation of VNN1 gene expression. In addition, the VNN1 3'UTR contains a sequence that is completely complementary to nucleotides 1 to 7 of miRNA-181a-5p. Overexpression of miR-181a-5p significantly decreased the expression level of VNN1 mRNA.
Conclusion
This study demonstrates that PPARα is an important transcriptional activator of VNN1 gene expression and that miRNA-181a-5p acts as a negative regulator of VNN1 expression in chicken hepatocytes.
Keywords: Chicken; Vanin1 Gene; PPARα; MiRNA-181a-5p; Regulatory Mechanism
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