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Updated: Oct 26, 2025

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA
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Knockdown of CNN3 Impairs Myoblast Proliferation, Differentiation, and Protein Synthesis via the mTOR Pathway

Yanling She1, Cheng Li1, Ting Jiang2

  • 1Guangdong Traditional Medical and Sports Injury Rehabilitation Research Institute, Guangdong Second Provincial General Hospital, Guangzhou, China.

Frontiers in Physiology
|July 26, 2021
PubMed
Abstract

Insights

Calponin 3 (CNN3) knockdown impairs muscle cell growth and differentiation by affecting AKT/mTOR and AMPK/mTOR signaling. Inhibiting CNN3 also impacts pathways involved in inflammation and cell death.

Area of Science:

  • Muscle biology and cell signaling
  • Molecular mechanisms of myogenesis
  • Cellular regulation of muscle regeneration

Background:

  • Myogenesis, the process of muscle formation, relies on intricate cell signaling.
  • Calponin 3 (CNN3) is implicated in muscle differentiation and regeneration, but its exact role is unclear.
  • This study investigates CNN3's function in C2C12 mouse muscle cells using a knockdown model.

Purpose of the Study:

  • To elucidate the role of Calponin 3 (CNN3) in regulating myoblast proliferation, migration, and differentiation.
  • To explore the involvement of the mTOR signaling pathway in CNN3-mediated myogenesis.
  • To identify downstream pathways affected by CNN3 modulation in muscle cells.

Main Methods:

  • Assessed myoblast proliferation, migration, differentiation, fusion, and protein synthesis in CNN3 knockdown C2C12 cells.
  • Investigated the AKT/mTOR and AMPK/mTOR pathways using an mTOR activator (MHY1485).
  • Utilized RNA sequencing, Gene Ontology (GO), KEGG, and Gene Set Enrichment Analysis (GSEA) to analyze regulatory mechanisms.

Main Results:

  • CNN3 knockdown reduced C2C12 cell proliferation and migration.
  • Myogenic differentiation, fusion, and protein synthesis were inhibited in CNN3 knockdown cells, with effects reversed by MHY1485.
  • KEGG and GSEA revealed alterations in the NOD-like receptor signaling pathway.

Conclusions:

  • CNN3 promotes C2C12 cell growth via regulation of AKT/mTOR and AMPK/mTOR signaling pathways.
  • CNN3 inhibition may activate pathways related to necroptosis, apoptosis, and inflammation, including the NOD-like receptor pathway.
Keywords:
C2C12 myoblastsCNN3differentiationmTOR pathwayproliferationprotein synthesis

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