中国农业科学 ›› 2025, Vol. 58 ›› Issue (8): 1638-1649.doi: 10.3864/j.issn.0578-1752.2025.08.014

• 畜牧·兽医 • 上一篇    下一篇

SCD5通过TRIM15调控脂滴数量的机制研究

白文哲1,2(), 李继豪1, 方钱海1,2, 张帆1,2, 胡睿祺1, 陈洪波2, 毕延震1(), 王锐2()   

  1. 1 湖北省农业科学院畜牧兽医研究所/动物胚胎工程及分子育种湖北省重点实验室,武汉 430064
    2 武汉轻工大学动物科学与营养工程学院,湖北省家畜种业技术创新中心,武汉 430023
  • 收稿日期:2024-10-15 接受日期:2025-02-28 出版日期:2025-04-16 发布日期:2025-04-21
  • 通信作者:
    毕延震,E-mail:
    王锐,E-mail:
  • 联系方式: 白文哲,Tel:15926607606;E-mail:baiwenzhe1@126.com。
  • 基金资助:
    农业生物育种重大项目(2023ZD0404703); 国家自然科学基金(32472882); 湖北省自然科学基金创新群体项目(2024AFA027); 湖北省重点研发计划(2023BBB032); 湖北省农业关键核心技术攻关项目(HBNYHXGG2023-7); 湖北省生猪种业高质量发展项目(HBZY2023B006-04)

Mechanistic Study of SCD5 Regulation of Lipid Droplet Quantity via TRIM15

BAI WenZhe1,2(), LI JiHao1, FANG QianHai1,2, ZHANG Fan1,2, HU RuiQi1, CHEN HongBo2, BI YanZhen1(), WANG Rui2()   

  1. 1 Key Laboratory of Animal Embryo Engineering and Molecular Breeding in Hubei Province, Institute of Animal Husbandry and Veterinary Medicine, Hubei Academy of Agricultural Sciences, Wuhan 430064
    2 Hubei Provincial Livestock Seed Industry Technology Innovation Center, Schoool of Animal Science and Nutritional Engineering, Wuhan Polytechnic University, Wuhan 430023
  • Received:2024-10-15 Accepted:2025-02-28 Published:2025-04-16 Online:2025-04-21

摘要:

【背景】脂肪沉积对猪肉的风味、嫩度和色泽有着重要影响,硬脂酰辅酶A去饱和酶(stearoyl-CoA desaturase, SCD)在调控脂质代谢过程中发挥关键作用,其中家族成员硬脂酰辅酶A去饱和酶-5(stearoyl-CoA desaturase 5, SCD5)作为单不饱和脂肪酸(monounsaturated fatty acid, MUFA)限速酶,调控单不饱和脂肪酸组成与三酰基甘油(TAG)含量。然而SCD5在脂滴(Lipid droplet, LD)生物发生过程中具体影响尚不明确。三元基序蛋白家族成员15(Tripartite Motif Containing 15, TRIM15)通过调节TAG代谢影响脂滴生物发生,并且在SCD5缺失后其表达量显著下降,两者之间的调控关系及其在脂滴形成过程中的作用机制仍有待深入探讨。【目的】探究SCD5对脂滴生物发生的调控作用以及SCD5是否通过TRIM15介导脂滴的形成与积累,进一步揭示SCD5在脂质代谢中的作用机制,为猪肉品质性状的遗传改良提供理论支持。【方法】本研究通过猪肾细胞(porcine kidney-15, PK-15)系利用CRISPR/Cas9 技术构建PK-15 SCD5敲除细胞系,通过转染SCD5过表达载体构建PK-15 SCD5过表达细胞系,以及在小鼠成肌细胞(C2C12)中过表达SCD5。利用Bodipy染色流式细胞术分析脂滴含量;利用Bodipy染色、共聚焦成像和油红O染色观察脂滴数量,探究SCD5对脂滴生物发生过程的作用。随后对PK-15 SCD5缺失细胞进行RNA-seq测序发现TRIM15显著下调,通过RT-qPCR和Western blot验证TRIM15的表达量,并构建TRIM15过表达载体在PK-15 SCD5缺失细胞中过表达TRIM15,探讨SCD5TRIM15在LD生物发生过程中的作用。【结果】Bodipy染色流式细胞术结果表明在PK-15 SCD5缺失细胞中脂滴含量显著降低,过表达SCD5显著增加脂滴含量;在C2C12中过表达SCD5结果相同。Bodipy染色共聚焦成像和油红O染色结果显示,SCD5缺失显著降低了PK-15细胞LD的数量而过表达SCD5后LD的数量显著增加;在C2C12细胞中异位表达SCD5则结果与之相同;对PK-15 SCD5缺失细胞进行RNA-seq测序后分析,GO和KEGG富集分析揭示SCD5与多细胞生物发育和脂质代谢途径相关,表明SCD5调控脂质代谢。通过对调控脂质代谢的基因进行分析发现TRIM15下调最为显著,这表明SCD5可能通过TRIM15调控脂质代谢。在PK-15 SCD5缺失细胞中过表达TRIM15,Bodipy染色流式细胞术以及Bodipy染色和油红O染色结果表明TRIM15增加了PK-15 SCD5缺失细胞的脂滴含量同时PK-15 SCD5缺失细胞中的LD数量显著增加。【结论】SCD5通过调控TRIM15的表达来介导LD生物发生进而调控LD的数量,调控脂肪沉积,这为猪肉品质改良提供了理论支持。

关键词: SCD5, TRIM15, 脂滴, 脂质代谢

Abstract:

【Background】 Fat deposition plays a critical role in determining pork quality traits such as flavor, tenderness, and color. Stearoyl-CoA desaturase (SCD) is a key enzyme involved in the regulation of lipid metabolism. Among its family members, stearoyl-CoA desaturase 5 (SCD5) functions as a rate-limiting enzyme for the synthesis of monounsaturated fatty acids (MUFAs), thereby influencing fatty acid composition and triacylglycerol (TAG) content. However, the specific role of SCD5 in the biogenesis of lipid droplets (LD) remains unclear. Tripartite motif-containing 15 (TRIM15), a member of the tripartite motif protein family, has been shown to affect LD biogenesis by modulating TAG metabolism. Notably, the expression of TRIM15 is significantly downregulated upon SCD5 deletion, suggesting a potential regulatory relationship between the two. Nevertheless, the exact mechanism of their interaction during LD formation remains to be elucidated.【Objective】This study aims to investigate the regulatory role of SCD5 in LD biogenesis and to determine whether SCD5 mediates LD formation and accumulation through TRIM15. The goal is to further elucidate the molecular mechanisms by which SCD5 regulates lipid metabolism, thereby providing a theoretical basis for the genetic improvement of pork quality traits.【Method】In this study, we utilized CRISPR/Cas9 technology to construct an SCD5 knockout cell line from porcine kidney cells (PK-15). We also established an SCD5-overexpressing PK-15 cell line by transfecting an SCD5 overexpression vector and overexpressed SCD5 in mouse adult myoblasts (C2C12). Lipid droplet content was analyzed via flow cytometry with Bodipy staining, and the number of lipid droplets was assessed using Bodipy staining, confocal microscopy, and oil red O staining to study SCD5's role in lipid droplet biogenesis. RNA-seq analysis of PK-15 SCD5-deficient cells showed significant downregulation of TRIM15. The expression of TRIM15 was confirmed by RT-qPCR and Western blot. To further investigate the roles of SCD5 and TRIM15 in lipid droplet biogenesis, a TRIM15 overexpression vector was constructed and introduced into PK-15 SCD5-deficient cells, exploring the role of SCD5 and TRIM15 in LD biogenesis.【Result】Flow cytometry analysis with Bodipy staining demonstrated that SCD5 deletion significantly reduced lipid droplet content, whereas SCD5 overexpression significantly increased it in PK-15 cells. Similar results were observed with SCD5 overexpression in C2C12 cells. Confocal imaging with Bodipy staining and oil red O staining revealed that SCD5 deletion significantly reduced the number of lipid droplets (LDs) in PK-15 cells. Overexpression of SCD5 in C2C12 cells produced similar results. RNA-seq analysis of PK-15 SCD5-deficient cells, combined with GO and KEGG enrichment analyses, indicated that SCD5 is involved in multicellular development and lipid metabolism pathways, suggesting its regulatory role in lipid metabolism. Gene analysis related to lipid metabolism identified TRIM15 as the most significantly downregulated gene, suggesting that SCD5 may regulate lipid metabolism via TRIM15. Overexpression of TRIM15 in SCD5-deficient PK-15 cells was assessed using Bodipy-stained flow cytometry, confocal imaging, and oil red O staining. The results showed that TRIM15 overexpression restored lipid droplet content and significantly increased the number of LDs in SCD5-deficient cells.【Conclusion】In summary, this study demonstrated that SCD5 mediates LD biogenesis and regulates LD quantity by modulating TRIM15 expression, thereby controlling fat deposition. These findings provide theoretical support for improving pork quality.

Key words: SCD5, TRIM15, Lipid droplets, Lipid metabolism