AccScience Publishing / GPD / Online First / DOI: 10.36922/gpd.2991
Submit to GPD
Cite this article
54
Download
1142
Views
Journal Browser
Volume | Year
Issue
Search
Forthcoming Issue
Current Issue
View All
News and Announcements
View All
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*
Show Less
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/ )
Download PDF
Cite
XML
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.
References
  1. McPherron AC, Lawler AM, Lee SJ. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature. 1997;387(6628):83-90. doi: 10.1038/387083a0

 

  1. Weng S, Zhao Y, Yu C, et al. Construction of a rAAV-SaCas9 system expressing eGFP and its application to improve muscle mass. Biotechnol Lett. 2021;43(11):2111-2129. doi: 10.1007/s10529-021-03183-13

 

  1. McPherron AC, Lee SJ. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci U S A. 1997;94(23):12457-12461. doi: 10.1073/pnas.94.23.12457

 

  1. Grobet L, Martin LJ, Poncelet D, et al. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat Genet. 1997;17(1):71-74. doi: 10.1038/ng0997-71

 

  1. Clop A, Marcq F, Takeda H, et al. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nat Genet. 2006;38(7):813-818. doi: 10.1038/ng18106

 

  1. Mosher DS, Quignon P, Bustamante CD, et al. A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs. PLoS Genet. 2007;3(5):e79. doi: 10.1371/journal.pgen.0030079

 

  1. Wang K, Tang X, Xie Z, et al. CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs. Transgenic Res. 2017;26(6):799-805. doi: 10.1007/s11248-017-0044-z

 

  1. Schuelke M, Wagner KR, Stolz LE, et al. Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med. 2004;350(26):2682-2688. doi: 10.1056/NEJMoa040933

 

  1. Cui Y, Yi Q, Sun W, et al. Molecular basis and therapeutic potential of myostatin on bone formation and metabolism in orthopedic disease. Biofactors. 2023;49(1):21-31. doi: 10.1002/biof.1675

 

  1. Cai C, Qian L, Jiang S, et al. Loss-of-function myostatin mutation increases insulin sensitivity and browning of white fat in Meishan pigs. Oncotarget. 2017;8(21):34911-34922. doi: 10.18632/oncotarget.16822

 

  1. Lee SJ. Targeting the myostatin signaling pathway to treat muscle loss and metabolic dysfunction. J Clin Invest. 2021;131(9):e148372. doi: 10.1172/JCI148372

 

  1. El Shafey N, Guesnon M, Simon F, et al. Inhibition of the myostatin/Smad signaling pathway by short decorin-derived peptides. Exp Cell Res. 2016;341(2):187-195. doi: 10.1016/j.yexcr.2016.01.019

 

  1. Sharma M, McFarlane C, Kambadur R, Kukreti H, Bonala S, Srinivasan S. Myostatin: Expanding horizons. IUBMB Life. 2015;67(8):589-600. doi: 10.1002/iub.1392

 

  1. Philip B, Lu Z, Gao Y. Regulation of GDF-8 signaling by the p38 MAPK. Cell Signal. 2005;17(3):365-375. doi: 10.1016/j.cellsig.2004.08.003

 

  1. Rao PK, Kumar RM, Farkhondeh M, Baskerville S, Lodish HF. Myogenic factors that regulate expression of muscle-specific microRNAs. Proc Natl Acad Sci U S A. 2006;103(23):8721-8726. doi: 10.1073/pnas.0602831103

 

  1. Liu N, Williams AH, Kim Y, et al. An intragenic MEF2- dependent enhancer directs muscle-specific expression of microRNAs 1 and 133. Proc Natl Acad Sci U S A. 2007;104(52):20844-20849. doi: 10.1073/pnas.0710558105

 

  1. Callis TE, Chen JF, Wang DZ. MicroRNAs in skeletal and cardiac muscle development. DNA Cell Biol. 2007;26(4):219-225. doi: 10.1089/dna.2006.0556

 

  1. van Rooij E, Liu N, Olson EN. MicroRNAs flex their muscles. Trends Genet. 2008;24(4):159-166. doi: 10.1016/j.tig.2008.01.007

 

  1. Rachagani S, Cheng Y, Reecy JM. Myostatin genotype regulates muscle-specific miRNA expression in mouse pectoralis muscle. BMC Res Notes. 2010;3:297. doi: 10.1186/1756-0500-3-297

 

  1. Wada S, Kato Y, Okutsu M, et al. Translational suppression of atrophic regulators by microRNA-23a integrates resistance to skeletal muscle atrophy. J Biol Chem. 2011;286(44):38456-38465. doi: 10.1074/jbc.M111.271270

 

  1. Weng S, Gao F, Wang J, et al. Improvement of muscular atrophy by AAV-SaCas9-mediated myostatin gene editing in aged mice. Cancer Gene Ther. 2020;27(12):960-975. doi: 10.1038/s41417-020-0178-722

 

  1. Li S, Sun Z, Chen T, et al. The role of miR-431-5p in regulating pulmonary surfactant expression in vitro. Cell Mol Biol Lett. 2019;24:25. doi: 10.1186/s11658-019-0150-4

 

  1. Giannesini B, Vilmen C, Amthor H, Bernard M, Bendahan D. Lack of myostatin impairs mechanical performance and ATP cost of contraction in exercising mouse gastrocnemius muscle in vivo. Am J Physiol Endocrinol Metab. 2013;305(1):E33-E40. doi: 10.1152/ajpendo.00651.201224

 

  1. Relizani K, Mouisel E, Giannesini B, et al. Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy. Mol Ther. 2014;22(8):1423-1433. doi: 10.1038/mt.2014.90

 

  1. Pearsall RS, Davies MV, Cannell M, et al. Follistatin-based ligand trap ACE-083 induces localized hypertrophy of skeletal muscle with functional improvement in models of neuromuscular disease. Sci Rep. 2019;9(1):11392. doi: 10.1038/s41598-019-47818-w

 

  1. LeBrasseur NK, Schelhorn TM, Bernardo BL, Cosgrove PG, Loria PM, Brown TA. Myostatin inhibition enhances the effects of exercise on performance and metabolic outcomes in aged mice. J Gerontol A Biol Sci Med Sci. 2009;64(9):940-948. doi: 10.1093/gerona/glp068

 

  1. Braga M, Pervin S, Norris K, Bhasin S, Singh R. Inhibition of in vitro and in vivo brown fat differentiation program by myostatin. Obesity (Silver Spring). 2013;21(6):1180-1188. doi: 10.1002/oby.20117

 

  1. Wang P, Liu Z, Zhang X, et al. CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells. Protein Cell. 2018;9(11):945-965. doi: 10.1007/s13238-018-0560-5

 

  1. Pascucci FA, Ladelfa MF, Toledo MF, Escalada M, Suberbordes M, Monte M. MageC2 protein is upregulated by oncogenic activation of MAPK pathway and causes impairment of the p53 transactivation function. Biochim Biophys Acta Mol Cell Res. 2021;1868(3):118918. doi: 10.1016/j.bbamcr.2020.118918

 

  1. Hitachi K, Nakatani M, Tsuchida K. Myostatin signaling regulates Akt activity via the regulation of miR-486 expression. Int J Biochem Cell Biol. 2014;47:93-103. doi: 10.1016/j.biocel.2013.12.003

 

  1. Wu R, Li H, Li T, Zhang Y, Zhu D. Myostatin regulates miR-431 expression via the Ras-Mek-Erk signaling pathway. Biochem Biophys Res Commun. 2015;461(2):224-229. doi: 10.1016/j.bbrc.2015.03.150
Share
Back to top
Gene & Protein in Disease, Electronic ISSN: 2811-003X Published by AccScience Publishing
We use cookies on our website to ensure you get the best experience.
Read more about our cookies here
Accept