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CRISPR/Cas9介导的来航鸡雄性肝癌细胞系中Vanin-1基因敲除及其对脂质代谢的影响。

CRISPR/Cas9-mediated knockout of the Vanin-1 gene in the Leghorn Male Hepatoma cell line and its effects on lipid metabolism.

作者信息

Xu Lu, Wang Zhongliang, Liu Shihao, Wei Zhiheng, Yu Jianfeng, Li Jun, Li Jie, Yao Wen, Gu Zhiliang

机构信息

School of Biology and Food Engineering, Changshu Institute of Technology, Changshu, 215500, China.

College of Animal Science & Technology, Nanjing Agriculture University, Nanjing, 210000, China.

出版信息

Anim Biosci. 2024 Mar;37(3):437-450. doi: 10.5713/ab.23.0162. Epub 2023 Nov 1.

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.

摘要

目的

泛酰巯基乙胺酶1(VNN1)是一种催化泛酰巯基乙胺水解生成泛酸和半胱胺的泛肽酶。我们之前的研究表明,VNN1在鸡肝脏中特异性表达,且受到微小RNA-122的负调控。然而,VNN1在鸡肝脏脂质代谢中的功能尚未阐明。

方法

首先,我们检测了禁食24小时的4周龄鸡中VNN1 mRNA的表达。接下来,通过CRISPR/Cas9系统在鸡来航鸡雄性肝癌细胞系中敲除VNN1。在来航鸡雄性肝癌细胞系中敲除VNN1后,通过油红染色检测脂质沉积,并分析甘油三酯(TG)、低密度脂蛋白-C(LDL-C)和高密度脂蛋白-C(HDL-C)的含量。然后,我们通过RNA测序捕获了VNN1修饰的LMH细胞与原始LMH细胞之间的各种差异表达基因(DEG)。

结果

首先,禁食诱导VNN1表达。同时,我们成功利用CRISPR/Cas9系统在鸡LMH细胞系中实现了VNN1的靶向突变。此外,与野生型LMH细胞相比,LMH-KO-VNN1细胞中VNN1 mRNA的表达水平降低(p<0.0001)。与对照组相比,通过油红染色发现敲除VNN1后脂质沉积减少,同时,LMH-KO-VNN1细胞中TG和LDL-C的含量显著降低,HDL-C的含量增加。转录组测序显示,LMH-KO-VNN1细胞与原始LMH细胞之间有1335个DEG。在这些DEG中,431个上调,904个下调。对所有DEG进行基因本体分析表明,脂肪酸生物合成和长链脂肪酸生物合成等脂质代谢相关途径得到富集。KEGG通路分析表明,“脂质代谢途径”“能量代谢”和“碳水化合物代谢”得到富集。共有76个DEG参与这些途径,其中29个基因上调(如细胞色素P450家族7亚家族A成员1、ELOVL脂肪酸延长酶2和载脂蛋白A4),47个基因下调(如磷酸烯醇式丙酮酸羧激酶1),这是由于在LMH细胞中敲除VNN1所致。

结论

这些结果表明,VNN1在协调鸡肝脏脂质代谢中起重要作用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c070/10915194/de236f4abf51/ab-23-0162f1.jpg

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