PFN1-PTEN通过调控PI3K/AKT/mTOR抑制牛骨骼肌卫星细胞分化

doi:10.11843/j.issn.0366-6964.2025.11.012

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (11): 5489-5501.doi: 10.11843/j.issn.0366-6964.2025.11.012

PFN1-PTEN通过调控PI3K/AKT/mTOR抑制牛骨骼肌卫星细胞分化

张帅(), 徐景(), 杨培鸿, 郭益文, 胡德宝, 李新, 丁向彬, 郭宏, 张林林*()

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 300384

收稿日期:2025-03-27 出版日期:2025-11-23 发布日期:2025-11-27
通讯作者: 张林林 E-mail:shuaizhang202202@163.com;Xj13662031206@163.com;zhanglinlin@tjau.edu.cn
作者简介:张帅(2000-)，女，河北邢台人，硕士，主要从事动物遗传育种研究，E-mail: shuaizhang202202@163.com
徐景(1995-)，女，四川眉山人，硕士，主要从事动物遗传育种研究，E-mail: Xj13662031206@163.com
第一联系人：
张帅和徐景为同等贡献作者
基金资助:
天津市畜禽分子育种与生物技术重点实验室开放课题基金(XM202502)；国家自然科学基金青年基金(31902151)

PFN1-PTEN Inhibits Bovine Skeletal Muscle Satellite Cell Differentiation by Regulating the PI3K/AKT/mTOR Pathway

ZHANG Shuai(), XU Jing(), YANG Peihong, GUO Yiwen, HU Debao, LI Xin, DING Xiangbin, GUO Hong, ZHANG Linlin*()

Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Farming, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384, China

Received:2025-03-27 Online:2025-11-23 Published:2025-11-27
Contact: ZHANG Linlin E-mail:shuaizhang202202@163.com;Xj13662031206@163.com;zhanglinlin@tjau.edu.cn

摘要/Abstract

摘要：

旨在探究PFN1通过与PTEN互作对牛骨骼肌卫星细胞(bovine skeletal muscle satellite cells, BSMSCs)成肌分化的影响。本试验将小片段干扰RNA(si-RNA)与过表达质粒分别转染到胎牛腿肌剥离的原代骨骼肌卫星细胞中，每个处理设计3个生物学重复。利用光学显微镜观察经过不同处理细胞分化的情况，在分化期的特定时间点收取细胞RNA与蛋白样品，分别利用实时荧光定量PCR(qRT-PCR)以及蛋白免疫印迹(Western blot)进行分析。试验结果显示：通过光学显微镜观察发现，干扰PFN1后牛骨骼肌卫星细胞可以形成更多粗大的肌管；qRT-PCR和Western blot证实，细胞分化标志基因MyHC表达量显著增加；而过表达PFN1后牛骨骼肌卫星细胞形成的肌管较细、量少；qRT-PCR和Western blot证实，MyHC表达量显著降低。通过Co-IP试验证实PFN1可以与PTEN互作，过表达PFN1可显著提升PTEN蛋白水平。在光学显微镜下观察发现，干扰PTEN后牛骨骼肌卫星细胞形成的肌管很少；qRT-PCR和Western blot试验证实，MyHC、MyOG在mRNA水平表达量显著增加，MyHC在蛋白水平显著降低；PI3K/AKT/mTOR信号通路关键蛋白表达量显著下调但通路最终关键作用蛋白p-mTOR显著增加。干扰PFN1后PI3K/AKT/mTOR信号通路关键蛋白表达量都显著增加。过表达PFN1后PI3K/AKT/mTOR信号通路关键蛋白显著下调。PFN1通过与PTEN互作抑制PI3K/AKT/mTOR通路活性，进而负调控BSMSCs成肌分化。本研究为肌肉发育的分子调控网络提供了新见解。

关键词: 牛, 骨骼肌卫星细胞, PFN1, 成肌分化, PI3K/AKT/mTOR

Abstract:

This study aimed to investigate the effect of PFN1 on the myogenic differentiation of bovine skeletal muscle satellite cells (BSMSCs) through its interaction with PTEN. Small interfering RNA (siRNA) and overexpression plasmids were transfected into primary bovine skeletal muscle satellite cells isolated from fetal bovine leg muscles, with 3 biological replicates for each treatment. The differentiation process of the cells was observed under an optical microscope. RNA and protein samples were collected at specific time points during the differentiation phase for analysis by quantitative real-time PCR (qRT-PCR) and Western blot. PFN1 knockdown promoted the formation of more robust myotubes, with significantly increased MyHC mRNA and protein levels. Conversely, PFN1 overexpression reduced myotube formation (thinner and fewer) and significantly decreased MyHC expression. Co-IP confirmed the interaction between PFN1 and PTEN, and PFN1 overexpression increased PTEN protein levels. Under optical microscope observation, PTEN knockdown resulted in fewer myotubes, with elevated MyHC and MyOG mRNA levels but reduced MyHC protein levels. Key proteins in the PI3K/AKT/mTOR pathway were downregulated, while p-mTOR levels increased. PFN1 knockdown upregulated key proteins in the PI3K/AKT/mTOR pathway, whereas PFN1 overexpression downregulated them. PFN1 interacts with PTEN to inhibit the PI3K/AKT/mTOR pathway, thereby negatively regulating BSMSCs myogenic differentiation. This study provides new insights into the molecular regulatory network of muscle development.

Key words: bovine, skeletal muscle satellite cells, PFN1, myogenic differentiation, PI3K/AKT/mTOR

中图分类号:

S823.2

张帅, 徐景, 杨培鸿, 郭益文, 胡德宝, 李新, 丁向彬, 郭宏, 张林林. PFN1-PTEN通过调控PI3K/AKT/mTOR抑制牛骨骼肌卫星细胞分化[J]. 畜牧兽医学报, 2025, 56(11): 5489-5501.

ZHANG Shuai, XU Jing, YANG Peihong, GUO Yiwen, HU Debao, LI Xin, DING Xiangbin, GUO Hong, ZHANG Linlin. PFN1-PTEN Inhibits Bovine Skeletal Muscle Satellite Cell Differentiation by Regulating the PI3K/AKT/mTOR Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5489-5501.

图/表 11

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基因Gene	引物序列(5′→3′) Sequence	产物大小/bp Product size
PFN1 forward	TCGTGAACGGGCTAACACTT	171
PFN1 reverse	CTTTGCCCATCAGCAGGACT
GAPDH forward	GAAGGTCGGAGTGAACGGAT	91
GAPDH reverse	ATTGATGGCGACGATGTCCA
PTEN forward	TATTCTTCTCCCCATTCCGCTGC	174
PTEN reverse	AATGGCTCTGGACTTGGCGG
MyoG forward	GGCTGACAAATGCCAGACTATCC	140
MyoG reverse	TGGTCCCTTGCTTTATCTCCCT
MyHC forward	CTGGAATCCGGAGGCAGAA	105
MyHC reverse	TTTTCGAAGGTAGGGAGCGG

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PFN1-PTEN通过调控PI3K/AKT/mTOR抑制牛骨骼肌卫星细胞分化

PFN1-PTEN Inhibits Bovine Skeletal Muscle Satellite Cell Differentiation by Regulating the PI3K/AKT/mTOR Pathway

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