AccScience Publishing / GPD / Online First / DOI: 10.36922/gpd.2991
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ORIGINAL RESEARCH ARTICLE

Establishment of a myostatin gene-knockout C2C12 cell line and evaluation of related microRNA expression

Shaoting Weng1 ,  Kaiqi Lian1 ,  Kunpeng Zhang1 ,  Shengming Ma1 ,  Wenhui Zhang1 ,  Zhongyi Luo1 ,  Ruifeng Chen2 ,  Liqiang Wang2 ,  Sen Lin3 ,  Xinying Ji4,5* ,  Yao Wang1*
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1 Department of Biotechnology, Anyang Institute of Technology, Anyang, Henan, China
2 Department of Diagnostic, Anyang District Hospital of Puyang City, Anyang, Henan, China
3 Laboratory of Molecular Biology, Anyang Kindstar Global Medical Laboratory Ltd, Anyang, Henan Anyang Kindstar Global Medical Laboratory Ltd, Anyang, Henan, China
4 Department of Basic Medicine, Faculty of Basic Medical Subjects, Shu-Qing Medical College of Zhengzhou, Zhengzhou, Henan, China
5 Department of Medicine, Huaxian County People’s Hospital, Anyang, Henan, China
GPD 2024, 3(2), 2991 https://doi.org/10.36922/gpd.2991
Submitted: 21 February 2024 | Accepted: 22 April 2024 | Published: 5 June 2024
© 2024 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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Abstract

The strategy of blocking myostatin (MSTN) signal transduction has long been regarded as a promising approach in the treatment of patients with muscle loss. However, individuals taking blocking agents often encounter issues such as lack of strength, fatigue, and poor muscle proliferation due to muscle hypertrophy and the involvement of multiple receptors. To address these challenges, a series of experiments were conducted on a C2C12 cell line in this study. First, the pX601-SaCas9-sgRNA/puro vector carrying a Cas9-encoded gene was constructed and subsequently used to produce Mstn-knockout (Mstn-KO) C2C12 cell lines. The expression level of the MSTN protein and the growth characteristics of the cell lines were verified. Moreover, the expression of muscle growth-related microRNAs in the cell lines was analyzed through real-time polymerase chain reaction (PCR). The results indicate that we have successfully established a method for constructing Mstn-KO cell lines with stable passage. No expression of the MSTN protein and strong cell proliferation were observed in the cell lines. Moreover, real-time PCR experiments showed that the expression levels of miR-1, miR-431, miR-206, and miR-133a were significantly increased (P < 0.01), the expression level of miR-23a was significantly increased (P < 0.05), and the expression level of miR-486 was significantly decreased (P < 0.05). These findings indicate that multiple miRNAs are closely associated with MSTN regulation. This study lays the foundation for further investigation into the effects of the Mstn gene on the physiological function of myoblasts and the development of drugs that block the MSTN signaling pathway.

Keywords
Myostatin
Gene knockout
C2C12 cell line
MicroRNA
Muscle growth
Funding
This work was supported by the National Natural Science Foundation of China (Grant No. 2023YFD2400302), the Key Research and Development Program Project of Anyang (Grant No. 2023C01SF172), and the Postdoctoral Research Start-up Project of Anyang Institute of Technology (Grant No. BHJ2021003).
Conflict of interest
The authors declare no competing interests.
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Gene & Protein in Disease, Electronic ISSN: 2811-003X Published by AccScience Publishing
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