目的:Vanin-1(VNN1)是一种泛肽酶,可催化泛肽水解产生泛酸和半胱胺。我们先前的研究表明,VNN1在鸡肝中特异性表达,并受到microRNA-122的负调控。然而,VNN1在鸡肝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周龄鸡的VNN1mRNA表达。接下来,通过CRISPR/Cas9系统在鸡来角雄性肝癌细胞系中敲除VNN1。油红染色检测脂质沉积,分析甘油三酯(TG)的含量,低密度脂蛋白C(LDL-C),和高密度脂蛋白-C(HDL-C)后VNN1基因敲除在Leghorn男性肝癌细胞系中。然后,我们通过RNA-seq捕获了VNN1修饰的LMH细胞和原始LMH细胞之间的各种差异表达基因(DEGs)。
结果:首先,空腹诱导的VNN1表达。同时,我们成功地使用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VNN1mRNA的表达水平降低(p<0.0001)。与对照相比,通过油红染色敲除VNN1后脂质沉积减少,同时,TG和LDL-C含量显著降低,LMH-KO-VNN1细胞中HDL-C含量增加。转录组测序显示LMH-KO-VNN1细胞和原始LMH细胞之间有1,335个DEGs。在这些DEG中,431被上调,904被下调。所有DEGs的基因本体论分析表明,脂质代谢相关通路,如脂肪酸生物合成和长链脂肪酸生物合成,丰富了。KEGG途径分析表明,“脂质代谢途径”,“能量代谢”,和“碳水化合物代谢”被丰富。共有76个DEGs参与了这些途径,其中29个基因(如细胞色素P450家族7亚家族A成员1、ELOVL脂肪酸延伸酶2和载脂蛋白A4)在LMH细胞中通过VNN1敲除被下调(如磷酸烯醇丙酮酸羧激酶1)。
结论:这些结果表明,VNN1在协调鸡肝脂质代谢中起重要作用。
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.
CONCLUSIONS: These results suggest that VNN1 plays an important role in coordinating lipid metabolism in the chicken liver.