Identification of genes differentially expressed in myogenin knock-down bovine muscle satellite cells during differentiation through RNA sequencing analysis

• Published in: PloS one 2014-03, Vol.9 (3), p.e92447-e92447
• Main Authors: Lee, Eun Ju, Malik, Adeel, Pokharel, Smritee, Ahmad, Sarafraz, Mir, Bilal Ahmad, Cho, Kyung Hyun, Kim, Jihoe, Kong, Joon Chan, Lee, Dong-Mok, Chung, Ki Yong, Kim, Sang Hoon, Choi, Inho
• Format: Article
• Language: English
• Subjects: Analysis; Animal genetics; Animal sciences; Animals; Annotations; Biology and Life Sciences; Biotechnology; Cattle; Cell cycle; Cell differentiation; Cell Differentiation - genetics; Cell Differentiation - physiology; Cell fusion; Cells (biology); Cells, Cultured; Computer applications; Cytoskeleton; Cytosol; Deoxyribonucleic acid; Differentiation (biology); DNA; DNA biosynthesis; DNA replication; Gene expression; Gene sequencing; Genes; Genomes; Genomics; Growth factors; Immunohistochemistry; Laboratories; Matrix metalloproteinases; Mitosis; Morphogenesis; Muscles; Musculoskeletal system; Mutation; MyoD protein; Myogenesis; Myogenin; Myogenin - genetics; Myogenin - metabolism; Myotubes; Phosphatases; Phosphates; Phosphoprotein phosphatase; Polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic acid; RNA; RNA sequencing; Rodents; Satellite cells; Satellite Cells, Skeletal Muscle - metabolism; Sequence Analysis, RNA - methods; Skeletal muscle; Studies; Transcription factors; Zoology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0092447

The expression of myogenic regulatory factors (MRFs) consisting of MyoD, Myf5, myogenin (MyoG) and MRF4 characterizes various phases of skeletal muscle development including myoblast proliferation, cell-cycle exit, cell fusion and the maturation of myotubes to form myofibers. Although it is well known that the function of MyoG cannot be compensated for other MRFs, the molecular mechanism by which MyoG controls muscle cell differentiation is still unclear. Therefore, in this study, RNA-Seq technology was applied to profile changes in gene expression in response to MyoG knock-down (MyoGkd) in primary bovine muscle satellite cells (MSCs). About 61-64% of the reads of over 42 million total reads were mapped to more than 13,000 genes in the reference bovine genome. RNA-Seq analysis identified 8,469 unique genes that were differentially expressed in MyoGkd. Among these genes, 230 were up-regulated and 224 were down-regulated by at least four-fold. DAVID Functional Annotation Cluster (FAC) and pathway analysis of all up- and down-regulated genes identified overrepresentation for cell cycle and division, DNA replication, mitosis, organelle lumen, nucleoplasm and cytosol, phosphate metabolic process, phosphoprotein phosphatase activity, cytoskeleton and cell morphogenesis, signifying the functional implication of these processes and pathways during skeletal muscle development. The RNA-Seq data was validated by real time RT-PCR analysis for eight out of ten genes as well as five marker genes investigated. This study is the first RNA-Seq based gene expression analysis of MyoGkd undertaken in primary bovine MSCs. Computational analysis of the differentially expressed genes has identified the significance of genes such as SAP30-like (SAP30L), Protein lyl-1 (LYL1), various matrix metalloproteinases, and several glycogenes in myogenesis. The results of the present study widen our knowledge of the molecular basis of skeletal muscle development and reveal the vital regulatory role of MyoG in retaining muscle cell differentiation.