miR-145-5p靶向IGF1R介导AKT通路抑制猪骨骼肌卫星细胞增殖和分化

doi:10.11843/j.issn.0366-6964.2023.05.012

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (5): 1893-1904.doi: 10.11843/j.issn.0366-6964.2023.05.012

miR-145-5p靶向IGF1R介导AKT通路抑制猪骨骼肌卫星细胞增殖和分化

员佳乐¹, 刘畅¹, 黄晓宇¹, 刘巧霞², 史明月¹, 李文霞¹, 牛瑾¹, 王首元¹, 高鹏飞¹, 郭晓红¹, 李步高¹, 路畅^1*, 曹果清^1*

1. 山西农业大学动物科学学院, 太谷 030801;
2. 山西省畜牧技术推广服务中心, 太原 030001

收稿日期:2022-09-15 出版日期:2023-05-23 发布日期:2023-05-20
通讯作者: 曹果清,主要从事猪新品种(系)培育与推广和地方猪种特色基因挖掘与育种应用,E-mail:anniecao710502@aliyun.com;路畅,主要从事非编码RNA调控猪脂肪代谢与骨骼肌发育研究和畜禽多组学数据库的构建,E-mail:luchang@sxau.edu.cn
作者简介:员佳乐(1998-),女,山西运城人,硕士生,主要从事动物遗传育种与繁殖研究,E-mail:1784219176@qq.com
基金资助:
国家自然科学基金(31872336);三晋学者支持计划专项经费资助(2016;2017);山西农业大学科技创新基金项目(2020BQ56);山西省基础研究计划项目(20210302124639)

miR-145-5p Inhibits the Proliferation and Differentiation of Porcine Skeletal Muscle Satellite Cells by Targeting IGF1R-Mediated AKT Pathway

YUN Jiale¹, LIU Chang¹, HUANG Xiaoyu¹, LIU Qiaoxia², SHI Mingyue¹, LI Wenxia¹, NIU Jin¹, WANG Shouyuan¹, GAO Pengfei¹, GUO Xiaohong¹, LI Bugao¹, LU Chang^1*, CAO Guoqing^1*

1. College of Animal Science, Shanxi Agricultural University, Taigu 030801, China;
2. Shanxi Animal Husbandry Technology Extansion Service Center, Taiyuan 030001, China

Received:2022-09-15 Online:2023-05-23 Published:2023-05-20

摘要/Abstract

摘要： 旨在探讨miR-145-5p对猪骨骼肌卫星细胞增殖和分化的影响。本研究采用qRT-PCR检测miR-145-5p的组织表达谱和发育性表达规律;选用1 d晋汾白猪趾长伸肌进行骨骼肌卫星细胞的分离与培养,分别转染miR-145-5p模拟物(mimics)及其对照组(mimics NC),miR-145-5p抑制剂(inhibitor)及其对照组(inhibitor NC),每个处理3个重复,利用qRT-PCR、EdU和CCK-8方法检测增殖相关基因表达量、EdU阳性细胞数和细胞增殖活性;待猪骨骼肌卫星细胞分化后利用qRT-PCR、Western blot和免疫荧光染色方法探究分化相关基因mRNA和蛋白水平及肌管形成情况;采用双荧光素酶试验、qRT-PCR和Western blot探究miR-145-5p 对下游IGF1R和AKT通路的影响。结果显示,miR-145-5p基因在肝和肺中表达量最高,心和皮下脂肪中次之(P<0.05);随着日龄的增加,miR-145-5p在猪背最长肌中的表达量持续升高(P<0.05)。在猪骨骼肌卫星细胞体外培养过程中,转染miR-145-5p mimics极显著下调Ki67和CDK1的表达(P<0.01),显著下调PCNA和CDK4的表达(P<0.05),阳性细胞指数极显著降低(P<0.01),细胞活性显著低于对照组(P<0.05)。在分化方面,过表达miR-145-5p极显著下调MyOD、MyOG和Myf5的表达量(P<0.01),MyOD蛋白水平显著降低(P<0.05),MyHC阳性肌管面积少于对照组;转染miR-145-5p inhibitor则出现相反的结果。过表达miR-145-5p显著降低IGF1R mRNA和蛋白的相对表达量(P<0.05),显著降低AKT蛋白的磷酸化水平;而干扰miR-145-5p能极显著上调IGF1R mRNA和蛋白的相对表达量(P<0.01),并能挽救转染si-IGF1R对p-AKT蛋白的抑制作用(P<0.01)。本研究结果表明,miR-145-5p通过负调控IGF1R mRNA和蛋白的相对表达水平,并影响AKT通路,抑制猪骨骼肌卫星细胞的增殖和分化,进而参与骨骼肌的发育过程。研究结果丰富了猪肌肉生长发育的分子网络,并为肌肉性状的分子育种提供了靶标。

关键词: miR-145-5p, IGF1R, AKT通路, 增殖和分化, 骨骼肌卫星细胞

Abstract: This study aimed to investigate the effect of miR-145-5p on the proliferation and differtiation of porcine skeletal muscle satellite cells. The tissue expression profile and developmental expression pattern of miR-145-5p were detected by qRT-PCR. The skeletal muscle satellite cells were isolated and cultured from the extensor digitorum longus of one-day old Jinfen White pig. The miR-145-5p simulant (mimics) and its control group (mimics NC), miR-145-5p inhibitor (inhibitor) and its control group (inhibitor NC) were transfected respectively. Each treatment had three replicates. The proliferation-related genes, the number of EdU-positive cells and the proliferative activity of cells were detected by qRT-PCR, EdU and CCK-8 methods. After the differentiation of porcine skeletal muscle satellite cells, qRT-PCR, Western blot and immunofluorescence staining were used to investigate the level of mRNA and protein of differentiation related genes and the formation of myotubes. The effects of miR-145-5p on the downstream IGF1R and AKT pathways were investigated by double luciferase test, qRT-PCR and Western blot. The results showed that the expression of miR-145-5p was the highest in the liver and lung, followed by in heart and subcutaneous fat (P<0.05). The expression of miR-145-5p in the longissimus dorsi muscle continued to increase with the increase of age (P<0.05). During the culture procession of porcine skeletal muscle satellite cells in vitro, the expressions of Ki67 and CDK1 (P<0.01), and PCNA and CDK4 (P<0.05) were down-regulated significantly after transfection of miR-145-5p mimics. The positive cell index was very significantly decreased (P<0.01), and the cell viability was also significantly decreased (P<0.05). Regarding to the differentiation of porcine skeletal muscle satellite cells, the expressions of MyOD, MyOG and Myf5 were down-regulated significantly (P<0.01) by overexpression of miR-145-5p, the level of MyOD protein was inhibited (P<0.05), and the MyHC-positive myotube area was also less than that of the control group. Transfection of miR-145-5p inhibitor resulted in the opposite result. Overexpression of miR-145-5p significantly reduced the relative expression of IGF1R mRNA and protein(P<0.05), and significantly reduced the phosphorylation level of AKT protein. Interference with miR-145-5p increased significantly the relative expression of IGF1R mRNA and protein(P<0.01), and rescued the inhibitory effect of si-IGF1R on p-AKT protein(P<0.01). In conclusion, miR-145-5p negatively regulates the relative expression of IGF1R mRNA and protein, affects AKT pathway, inhibits the proliferation and differentiation of porcine skeletal muscle satellite cells, and then participates in the development of skeletal muscle. The results of this study enrich the molecular network of pig muscle growth and development, and provide molecular targets for molecular breeding of muscle traits.

Key words: miR-145-5p, IGF1R, AKT pathway, proliferation and differentiation, skeletal muscle satellite cells

中图分类号:

S828.2

员佳乐, 刘畅, 黄晓宇, 刘巧霞, 史明月, 李文霞, 牛瑾, 王首元, 高鹏飞, 郭晓红, 李步高, 路畅, 曹果清. miR-145-5p靶向IGF1R介导AKT通路抑制猪骨骼肌卫星细胞增殖和分化[J]. 畜牧兽医学报, 2023, 54(5): 1893-1904.

YUN Jiale, LIU Chang, HUANG Xiaoyu, LIU Qiaoxia, SHI Mingyue, LI Wenxia, NIU Jin, WANG Shouyuan, GAO Pengfei, GUO Xiaohong, LI Bugao, LU Chang, CAO Guoqing. miR-145-5p Inhibits the Proliferation and Differentiation of Porcine Skeletal Muscle Satellite Cells by Targeting IGF1R-Mediated AKT Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 1893-1904.

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miR-145-5p靶向IGF1R介导AKT通路抑制猪骨骼肌卫星细胞增殖和分化

miR-145-5p Inhibits the Proliferation and Differentiation of Porcine Skeletal Muscle Satellite Cells by Targeting IGF1R-Mediated AKT Pathway

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