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

Abstract

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

CLC Number:

S828.2

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

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