UCH-L3通过影响RAD51调控牛骨骼肌卫星细胞增殖与成肌分化

doi:10.19640/j.cnki.jtau.2023.02.001

天津农学院学报 ›› 2023, Vol. 30 ›› Issue (2): 1-6.doi: 10.19640/j.cnki.jtau.2023.02.001

• 研究与简报 • 下一篇

UCH-L3通过影响RAD51调控牛骨骼肌卫星细胞增殖与成肌分化

胡蕾, 王轶敏, 张林林, 李新, 郭益文, 丁向彬^通信作者

天津农学院动物科学与动物医学学院天津市农业动物繁育与健康养殖重点实验室,天津 300392

收稿日期:2021-08-23 出版日期:2023-04-30 发布日期:2023-06-28
通讯作者: 丁向彬（1978—）,男,教授,博士,研究方向为动物细胞工程与繁育生物技术。E-mail：xiangbinding@163.com。
作者简介:胡蕾（1995—）,女,硕士在读,研究方向为动物胚胎与转基因工程。E-mail：18888108159@163.com。
基金资助:
天津市自然科学基金项目（18JCYBJC29700）; 国家自然科学基金项目（311772559）

UCH-L3 regulating the proliferation and differentiation of bovine skeletal muscle satellite cells by influencing RAD51

Hu Lei, Wang Yimin, Zhang Linlin, Li Xin, Guo Yiwen, Ding Xiangbin^{Corresponding Author}

Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China

Received:2021-08-23 Online:2023-04-30 Published:2023-06-28

摘要/Abstract

摘要： 本研究旨在探究泛素蛋白酶体系统中羧基末端水解酶L3（Ubiquitin C-Terminal Hydrolase L3,UCH-L3）通过影响RAD51调控牛骨骼肌卫星细胞增殖与成肌分化的机制。使用已经建立的牛骨骼肌卫星细胞体外成肌分化模型,体外模拟骨骼肌的发育过程,采用免疫沉淀技术检测UCH-L3与RAD51是否相互作用,检测UCH-L3是否调控RAD51的泛素化水平,设计并合成RAD51的si-RNA,转染牛骨骼肌卫星细胞,筛选出有显著干扰效果的si-RNA,研究干扰RAD51后增殖和分化标志基因PAX7和MyHC表达水平变化,综合分析UCH-L3通过影响RAD51调控牛骨骼肌卫星细胞增殖与成肌分化的机制。结果表明：过表达UCH-L3后RAD51的蛋白水平显著升高,UCH-L3可以调控RAD51的表达水平。UCH-L3与RAD51存在体内相互作用,UCH-L3可以影响RAD51的蛋白稳定性,干扰UCH-L3表达可以缩短RAD51的半衰期,UCH-L3能够调控RAD51泛素化水平,干扰RAD51后,牛骨骼肌卫星细胞增殖标志因子PAX7的蛋白水平与对照组无差异,分化标志因子MyHC的蛋白水平显著低于对照组（P<0.05）,干扰RAD51表达对细胞增殖没有明显影响,但可以抑制牛骨骼肌卫星细胞的成肌分化。本研究结果表明,UCH-L3可以通过影响RAD51调控牛骨骼肌卫星细胞增殖与成肌分化进程。

关键词: RAD51, UCH-L3, 牛骨骼肌卫星细胞, 增殖, 成肌分化

Abstract: The purpose of this study was to explore the mechanism of carboxyl terminal hydrolase L3（UCH-L3）in ubiquitin proteasome system regulating the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells by affecting RAD51. Using the established in vitro myogenic differentiation model of bovine skeletal muscle satellite cells, this study simulated the development process of skeletal muscle in vitro, detected whether uch-l3 interacts with RAD51 by immunoprecipitation technology, and whether uch-l3 regulates the ubiquitination level of RAD51, designed and synthesized si-RNA of RAD51, transfected bovine skeletal muscle satellite cells, and screened si-RNA with significant interference effect, detected the changes of the expression levels of proliferation and differentiation marker genes Pax7 and MyHC after interfering with Rad51, and comprehensively analyzed the mechanism of UCH-L3 regulating the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells by affecting Rad51. The results showed that the protein levels of RAD51 increased significantly after over-expression of UCH-L3, and UCH-L3 could regulate the expression level of RAD51. UCH-L3 interacts with RAD51 in vivo. UCH-L3 can affect the protein stability of RAD51. Interfering with UCH-L3 expression can shorten the half-life of RAD51. UCH-L3 can regulate the ubiquitination level of RAD51. After interfering with RAD51, the protein level of Pax7, a marker of bovine skeletal muscle satellite cell proliferation, was not different from that of the control group, and the protein level of MyHC, a marker of differentiation, was significantly lower than that of the control group（P<0.05）. Interfering with the expression of RAD51 had no significant effect on cell proliferation, but could inhibit the myogenic differentiation of bovine skeletal muscle satellite cells. The results showed that UCH-L3 could regulate the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells by affecting RAD51.

Key words: RAD51, UCH-L3, bovine skeletal muscle satellite cells, proliferation, myogenic differentiation

中图分类号:

胡蕾, 王轶敏, 张林林, 李新, 郭益文, 丁向彬. UCH-L3通过影响RAD51调控牛骨骼肌卫星细胞增殖与成肌分化[J]. 天津农学院学报, 2023, 30(2): 1-6.

Hu Lei, Wang Yimin, Zhang Linlin, Li Xin, Guo Yiwen, Ding Xiangbin. UCH-L3 regulating the proliferation and differentiation of bovine skeletal muscle satellite cells by influencing RAD51[J]. Journal of Tianjin Agricultural University, 2023, 30(2): 1-6.

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http://xuebao.tjau.edu.cn/CN/Y2023/V30/I2/1

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UCH-L3通过影响RAD51调控牛骨骼肌卫星细胞增殖与成肌分化

UCH-L3 regulating the proliferation and differentiation of bovine skeletal muscle satellite cells by influencing RAD51

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