Page 114 - GPD-3-2

Gene & Protein in Disease

ORIGINAL RESEARCH ARTICLE
Establishment of a myostatin gene-knockout

C2C12 cell line and evaluation of related
microRNA expression

Shaoting Weng 1 , Kaiqi Lian , Kunpeng Zhang , Shengming Ma ,
1
1
1
1
1
Wenhui Zhang , Zhongyi Luo , Ruifeng Chen , Liqiang Wang , Sen Lin ,
2
3
2
1
Xinying Ji * , and Yao Wang *
4,5
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,
China 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
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
*Corresponding authors: conducted on a C2C12 cell line in this study. First, the pX601-SaCas9-sgRNA/puro vector
Xinying Ji carrying a Cas9-encoded gene was constructed and subsequently used to produce
(10190096@vip.henu.edu.cn) Mstn-knockout (Mstn-KO) C2C12 cell lines. The expression level of the MSTN protein
Yao Wang
(89742715@qq.com) 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-
Citation: Weng S, Lian K,
Zhang K, et al. Establishment of a time polymerase chain reaction (PCR). The results indicate that we have successfully
myostatin gene-knockout C2C12 established a method for constructing Mstn-KO cell lines with stable passage. No
cell line and evaluation of related expression of the MSTN protein and strong cell proliferation were observed in the cell
microRNA expression. Gene Protein
Dis. 2024;3(2):2991. lines. Moreover, real-time PCR experiments showed that the expression levels of miR-1,
doi: 10.36922/gpd.2991 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
Received: February 21, 2024
Accepted: April 22, 2024 miR-486 was significantly decreased (P < 0.05). These findings indicate that multiple
Published Online: June 5, 2024 miRNAs are closely associated with MSTN regulation. This study lays the foundation
Copyright: © 2024 Author(s). for further investigation into the effects of the Mstn gene on the physiological function
This is an Open-Access article of myoblasts and the development of drugs that block the MSTN signaling pathway.
distributed under the terms of the
Creative Commons Attribution
License, permitting distribution, Keywords: Myostatin; Gene knockout; C2C12 cell line; MicroRNA; Muscle growth
and reproduction in any medium,
provided the original work is
properly cited.
Publisher’s Note: AccScience
Publishing remains neutral with 1. Introduction
regard to jurisdictional claims in
published maps and institutional Myostatin (MSTN), a member of the transforming growth factor-β (TGF-β) superfamily,
1
affiliations. exerts a negative regulatory effect on skeletal muscle growth and development. Gene-
Volume 3 Issue 2 (2024) 1 doi: 10.36922/gpd.2991