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CRISPR/Cas9-mediated knockout of the Vanin-1 gene in the Leghorn Male Hepatoma cell line and its effects on lipid metabolism |
Lu Xu1,a 
, Zhongliang Wang1,2,a
, Shihao Liu1
, Zhiheng Wei1
, Jianfeng Yu1
, Jun Li1
, Jie Li1,2
, Wen Yao2
, Zhiliang Gu1,*
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1School of Biology and Food Engineering, Changshu Institute of Technology, Changshu, 215500, China
2College of Animal Science & Technology, Nanjing Agriculture University, Nanjing, 210000, China |
Correspondence:
Zhiliang Gu, Tel: +86-512-52251570, Fax: +86-512-52251562, Email: zhilianggu88@hotmail.com
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Received: 28 April 2023 • Revised: 12 June 2023 • Accepted: 18 September 2023
aThese authors contributed equally to this work. |
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| Abstract |
Objective
Vanin-1 (VNN1) is a pantetheinase that catalyses the hydrolysis of pantetheine
to produce pantothenic acid and cysteamine. Our previous studies have shown that the
VNN1 is specifically expressed in chicken liver which negatively regulated by microRNA-122.
However, the functions of the VNN1 in lipid metabolism in chicken liver haven’t been elucidated.
Methods
First, we detected the VNN1 mRNA expression in 4-week chickens which were fasted 24 hours. Next, knocked out VNN1 via CRISPR/Cas9 system in the chicken Leghorn Male Hepatoma cell line. Detected the lipid deposition via oil red staining and analysis the content of triglycerides (TG), low-density lipoprotein-C (LDL-C), and highdensity lipoprotein-C (HDL-C) after VNN1 knockout in Leghorn Male Hepatoma cell line. Then we captured various differentially expressed genes (DEGs) between VNN1-modified LMH cells and original LMH cells by RNA-seq.
Results
Firstly, fasting-induced expression of VNN1. Meanwhile, we successfully used the CRISPR/Cas9 system to achieve targeted mutations of the VNN1 in the chicken LMH cell line. Moreover, the expression level of VNN1 mRNA in LMH-KO-VNN1 cells decreased compared with that in the wild-type LMH cells (p<0.0001). Compared with control, lipid deposition was decreased after knockout VNN1 via oil red staining, meanwhile, the contents of TG and LDL-C were significantly reduced, and the content of HDL-C was increased in LMH-KO-VNN1 cells. Transcriptome sequencing showed that there were 1,335 DEGs between LMH-KO-VNN1 cells and original LMH cells. Of these DEGs, 431 were upregulated, and 904 were downregulated. Gene ontology analyses of all DEGs showed that the lipid metabolism-related pathways, such as fatty acid biosynthesis and long-chain fatty acid biosynthesis, were enriched. KEGG pathway analyses showed that “lipid metabolism pathway”, “energy metabolism”, and “carbohydrate metabolism” were enriched. A total of 76 DEGs were involved in these pathways, of which 29 genes were upregulated (such as cytochrome P450 family 7 subfamily A member 1, ELOVL fatty acid elongase 2, and apolipoprotein A4) and 47 genes were downregulated (such as phosphoenolpyruvate carboxykinase 1) by VNN1 knockout in the LMH cells.
Conclusion
These results suggest that VNN1 plays an important role in coordinating lipid metabolism in the chicken liver. |
| Keywords:
Chicken; CRISPR-Cas9; Liver Metabolism; RNA-seq; VNN1 |
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