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<p>Skeletal muscle development depends on the directed differentiation of myoblasts and their fusion into myotubes. Elucidating the mechanisms governing myoblast differentiation is essential for understanding muscle formation. Although suppressor of cytokine signaling 2 (<i>SOCS2</i>) has been implicated in this process, its precise regulatory role remains unclear. Here, the Cytosine Base Editor (CBE) system, offers a powerful approach for studying gene-specific functions, was used to investigate <i>SOCS2</i> specific functions. sgRNAs targeting the murine <i>SOCS2</i> gene were designed and expression plasmids were constructed. In C2C12 myoblasts, one sgRNA (sg1) mediated efficient base editing (53.0%), introducing a point mutation at amino acid 19 that generated a premature stop codon. Monoclonal cell lines with this mutation were established using limiting dilution. Western blot (WB) analysis confirmed a significant (<i>P</i> &lt; 0.01) reduction in SOCS2 protein expression in the edited cells, accompanied by elevated levels of Growth Hormone Receptor (GHR). Immunofluorescence (IF) staining further validated increased GHR expression following SOCS2 knockdown. Differentiation assays indicated that <i>SOCS2</i> knockout promoted C2C12 differentiation, with significantly (<i>P</i> &lt; 0.01) upregulated expression of the myogenic markers <i>MyoD1</i>, <i>MyoG</i> and <i>MYH1</i>. Proteomic sequencing revealed enrichment of differentially expressed proteins in the PI3K/AKT and mTOR signaling pathways. Correspondingly, WB results showed that SOCS2 knockout significantly (<i>P</i> &lt; 0.05) increased the expression of AKT, mTOR, and the phosphorylated forms of PI3K, AKT, and mTOR. Together, these findings demonstrate that CBE-mediated <i>SOCS2</i> knockout enhances C2C12 differentiation and activates the PI3K/AKT/mTOR signaling pathway, thereby contributing new insights into the molecular regulation of skeletal muscle development.</p>

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Cytosine base editor-mediated SOCS2 knockout promotes C2C12 cell differentiation via the PI3K/AKT/mTOR signaling pathway

  • 期刊论文

  • 发表日期：

    2025年11月6日

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  • Y. Wang,
  • C. J. Zhang,
  • Y. X. Li,
  • W. J. Liu,
  • P. Liu,
  • Y. P. Liu,
  • J. Zhang,
  • S. X. Cao,
  • H. X. Li

摘要

Skeletal muscle development depends on the directed differentiation of myoblasts and their fusion into myotubes. Elucidating the mechanisms governing myoblast differentiation is essential for understanding muscle formation. Although suppressor of cytokine signaling 2 (SOCS2) has been implicated in this process, its precise regulatory role remains unclear. Here, the Cytosine Base Editor (CBE) system, offers a powerful approach for studying gene-specific functions, was used to investigate SOCS2 specific functions. sgRNAs targeting the murine SOCS2 gene were designed and expression plasmids were constructed. In C2C12 myoblasts, one sgRNA (sg1) mediated efficient base editing (53.0%), introducing a point mutation at amino acid 19 that generated a premature stop codon. Monoclonal cell lines with this mutation were established using limiting dilution. Western blot (WB) analysis confirmed a significant (P < 0.01) reduction in SOCS2 protein expression in the edited cells, accompanied by elevated levels of Growth Hormone Receptor (GHR). Immunofluorescence (IF) staining further validated increased GHR expression following SOCS2 knockdown. Differentiation assays indicated that SOCS2 knockout promoted C2C12 differentiation, with significantly (P < 0.01) upregulated expression of the myogenic markers MyoD1, MyoG and MYH1. Proteomic sequencing revealed enrichment of differentially expressed proteins in the PI3K/AKT and mTOR signaling pathways. Correspondingly, WB results showed that SOCS2 knockout significantly (P < 0.05) increased the expression of AKT, mTOR, and the phosphorylated forms of PI3K, AKT, and mTOR. Together, these findings demonstrate that CBE-mediated SOCS2 knockout enhances C2C12 differentiation and activates the PI3K/AKT/mTOR signaling pathway, thereby contributing new insights into the molecular regulation of skeletal muscle development.