Animal Bioscience

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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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 Xu (School of Biology and Food Engineering, Changshu Institute of Technology) ;
  • Zhongliang Wang (School of Biology and Food Engineering, Changshu Institute of Technology) ;
  • Shihao Liu (School of Biology and Food Engineering, Changshu Institute of Technology) ;
  • Zhiheng Wei (School of Biology and Food Engineering, Changshu Institute of Technology) ;
  • Jianfeng Yu (School of Biology and Food Engineering, Changshu Institute of Technology) ;
  • Jun Li (School of Biology and Food Engineering, Changshu Institute of Technology) ;
  • Jie Li (School of Biology and Food Engineering, Changshu Institute of Technology) ;
  • Wen Yao (College of Animal Science & Technology, Nanjing Agriculture University) ;
  • Zhiliang Gu (School of Biology and Food Engineering, Changshu Institute of Technology)
  • Received : 2023.04.28
  • Accepted : 2023.09.18
  • Published : 2024.03.01
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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 high-density 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

Acknowledgement

We gratefully acknowledge the financial support for this study provided by the National Natural Science Foundation of China (31772593, 31472091, 31802050) and the Natural Science Foundation of Jiangsu Province (BK20191476).

References

  1. Wang SZ, Hu XX, Wang ZP, et al. Quantitative trait loci associated with body weight and abdominal fat traits on chicken chromosomes 3, 5 and 7. Genet Mol Res 2012;11:956-65. https://doi.org/10.4238/2012.April.19.1 
  2. Deeb N, Lamont SJ. Genetic architecture of growth and body composition in unique chicken populations. J Hered 2002;93:107-18. https://doi.org/10.1093/jhered/93.2.107 
  3. Li H, Deeb N, Zhou H, Mitchell AD, Ashwell CM, Lamont SJ. Chicken quantitative trait loci for growth and body composition associated with transforming growth factor-beta genes. Poult Sci 2003;82:347-56. https://doi.org/10.1093/ps/82.3.347 
  4. Le Mignon G, Desert C, Pitel F, et al. Using transcriptome profiling to characterize QTL regions on chicken chromosome 5. BMC Genomics 2009;10:575. https://doi.org/10.1186/1471-2164-10-575 
  5. Esau C, Davis S, Murray SF, et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab 2006;3:87-98. https://doi.org/10.1016/j.cmet.2006.01.005 
  6. Wang X, Shao F, Yu J, Jiang H, Gong D, Gu Z. MicroRNA-122 targets genes related to liver metabolism in chickens. Comp Biochem Physiol B Biochem Mol Biol 2015;184:29-35. https://doi.org/10.1016/j.cbpb.2015.02.002 
  7. Li Y, Wang X, Yu J, et al. MiR-122 targets the vanin 1 gene to regulate its expression in chickens. Poult Sci 2016;95:1145-50. https://doi.org/10.3382/ps/pew039 
  8. Wyse BW, Wittwer C, Hansen RG. Radioimmunoassay for pantothenic acid in blood and other tissues. Clin Chem 1979;25:108-11. https://doi.org/10.1093/clinchem/25.1.108 
  9. Jansen PA, Kamsteeg M, Rodijk-Olthuis D, et al. Expression of the vanin gene family in normal and inflamed human skin: induction by proinflammatory cytokines. J Investig Dermatol 2009;129:2167-74. https://doi.org/10.1038/jid.2009.67 
  10. Rommelaere S, Millet V, Gensollen T, et al. PPARalpha regulates the production of serum Vanin-1 by liver. FEBS Lett 2013;587:3742-8. https://doi.org/10.1016/j.febslet.2013.09.046 
  11. Van Diepen JA, Jansen PA, Ballak DB, et al. PPAR-alpha dependent regulation of vanin-1 mediates hepatic lipid metabolism. J Hepatol 2014;61:366-72.  https://doi.org/10.1016/j.jhep.2014.04.013
  12. Wang Z, Yu J, Hua N, et al. Regulation of chicken vanin1 gene expression by peroxisome proliferators activated receptor α and miRNA-181a-5p. Anim Biosci 2021;34:172-84. https://doi.org/10.5713/ajas.19.1000 
  13. Huang H, Dong X, Kang MX, et al. Novel blood biomarkers of pancreatic cancer-associated diabetes mellitus identified by peripheral blood-based gene expression profiles. Am J Gastroenterol 2010;105:1661-9. https://doi.org/10.1038/ajg.2010.32 
  14. Hsu P, Lander E, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014;157:1262-78. https://doi.org/10.1016/j.cell.2014.05.010 
  15. Yang D, Xu J, Zhu T, et al. Effective gene targeting in rabbits using RNA-guided Cas9 nucleases. J Mol Cell Biol 2014;6:97-9. https://doi.org/10.1093/jmcb/mjt047 
  16. Hai T, Teng F, Guo R, Li W, Zhou Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res 2014;24:372-5. https://doi.org/10.1038/cr.2014.11 
  17. Bai Y, He L, Li P, et al. Efficient genome editing in chicken DF-1 cells using the CRISPR/Cas9 system. G3 (Bethesda, Md.) 2016;6:917-23. https://doi.org/10.1534/g3.116.027706 
  18. Oishi I, Yoshii K, Miyahara D, Kagami H, Tagami T. Targeted mutagenesis in chicken using CRISPR/Cas9 system. Sci Rep 2016;6:23980. https://doi.org/10.1038/srep23980 
  19. Gandhi S, Piacentino ML, Vieceli FM, Bronner ME. Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo. Dev Biol 2017;432:86-97. https://doi.org/10.1016/j.ydbio.2017.08.036 
  20. Xiong Y, Xu Q, Lin S, Wang Y, Lin Y, Zhu J. Knockdown of LXRα inhibits goat intramuscular preadipocyte differentiation. Int J Mol Sci 2018;19:3037. https://doi.org/10.3390/ijms19103037 
  21. Dupre S, Graziani MT, Rosei MA, Fabi A, Del Grosso E. The enzymatic breakdown of pantethine to pantothenic acid and cystamine. Eur J Biochem 1970;16:571-8. https://doi.org/10.1111/j.1432-1033.1970.tb01119.x 
  22. Fu Y, Foden JA, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 2013;31:822-6. https://doi.org/10.1038/nbt.2623 
  23. Hsu P, Scott D, Weinstein J, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 2013;31:827-32. https://doi.org/10.1038/nbt.2647 
  24. Desert C, Duclos MJ, Blavy P, et al. Transcriptome profiling of the feeding-to-fasting transition in chicken liver. BMC Genomics 2008;9:611. https://doi.org/10.1186/1471-2164-9-611 
  25. Veron N, Qu Z, Kipen PA, Hirst CE, Marcelle C. CRISPR mediated somatic cell genome engineering in the chicken. Dev Biol 2015;407:68-74. https://doi.org/10.1016/j.ydbio.2015.08.007 
  26. Gregory MK, Geier MS, Gibson RA, James MJ. Functional characterization of the chicken fatty acid elongases. J Nutr 2013;143:12-6. https://doi.org/10.3945/jn.112.170290 
  27. Wang F, Kohan AB, Lo CM, Liu M, Howles P, Tso P. Apolipoprotein A-IV: a protein intimately involved in metabolism. J Lipid Res 2015;56:1403-18. https://doi.org/10.1194/jlr.R052753 
  28. Jacobo-Albavera L, Aguayo-de la Rosa PI, Villarreal-Molina T, et al. VNN1 gene expression levels and the G-137T polymorphism are associated with HDL-C levels in Mexican prepubertal children. PloS one 2012;7:e49818. https://doi.org/10.1371/journal.pone.0049818 
  29. Hu YW, Wu SG, Zhao JJ, et al. VNN1 promotes atherosclerosis progression in apoE(-/-) mice fed a high-fat/high-cholesterol diet. J Lipid Res 2016;57:1398-411. https://doi.org/10.1194/jlr.M065565 
  30. Li T, Matozel M, Boehme S, et al. Overexpression of cholesterol 7α-hydroxylase promotes hepatic bile acid synthesis and secretion and maintains cholesterol homeostasis. Hepatology 2011;53:996-1006. https://doi.org/10.1002/hep.24107 
  31. Lee MS, Park JY, Freake H, Kwun IS, Kim Y. Green tea catechin enhances cholesterol 7alpha-hydroxylase gene expression in HepG2 cells. Br J Nutr 2008;99:1182-5. https://doi.org/10.1017/s0007114507864816