Network Analysis for the Identification of Differentially Expressed Hub Genes Using Myogenin Knock-down Muscle Satellite Cells

PLoS One. 2015 Jul 22;10(7):e0133597. doi: 10.1371/journal.pone.0133597. eCollection 2015.

Abstract

Muscle, a multinucleate syncytium formed by the fusion of mononuclear myoblasts, arises from quiescent progenitors (satellite cells) via activation of muscle-specific transcription factors (MyoD, Myf5, myogenin: MYOG, and MRF4). Subsequent to a decline in Pax7, induction in the expression of MYOG is a hallmark of myoblasts that have entered the differentiation phase following cell cycle withdrawal. It is evident that MYOG function cannot be compensated by any other myogenic regulatory factors (MRFs). Despite a plethora of information available regarding MYOG, the mechanism by which MYOG regulates muscle cell differentiation has not yet been identified. Using an RNA-Seq approach, analysis of MYOG knock-down muscle satellite cells (MSCs) have shown that genes associated with cell cycle and division, DNA replication, and phosphate metabolism are differentially expressed. By constructing an interaction network of differentially expressed genes (DEGs) using GeneMANIA, cadherin-associated protein (CTNNA2) was identified as the main hub gene in the network with highest node degree. Four functional clusters (modules or communities) were identified in the network and the functional enrichment analysis revealed that genes included in these clusters significantly contribute to skeletal muscle development. To confirm this finding, in vitro studies revealed increased expression of CTNNA2 in MSCs on day 12 compared to day 10. Expression of CTNNA2 was decreased in MYOG knock-down cells. However, knocking down CTNNA2, which leads to increased expression of extracellular matrix (ECM) genes (type I collagen α1 and type I collagen α2) along with myostatin (MSTN), was not found significantly affecting the expression of MYOG in C2C12 cells. We therefore propose that MYOG exerts its regulatory effects by acting upstream of CTNNA2, which in turn regulates the differentiation of C2C12 cells via interaction with ECM genes. Taken together, these findings highlight a new mechanism by which MYOG interacts with CTNNA2 in order to promote myoblast differentiation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Cell Line
  • Gene Expression Regulation*
  • Gene Knockdown Techniques*
  • Gene Regulatory Networks*
  • Humans
  • Myogenin / genetics*
  • Myogenin / metabolism
  • Satellite Cells, Skeletal Muscle / cytology
  • Satellite Cells, Skeletal Muscle / metabolism*

Substances

  • MYOG protein, human
  • Myogenin

Grants and funding

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Republic of Korea government (MSIP; no. 2014R1A2A2A01006324) along with the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2059308).