Action mechanism of myogenetic oligodeoxynucleotides (myoDNs)

Action mechanism of myogenetic oligodeoxynucleotides (myoDNs)

Sayaka Shinji, Tomohide Takaya.

Department of Agriculture, Graduate School of Science and Technology, Shinshu University, Japan.

International Joint Seminar 2019 Sejong University-Shinshu University-Kasetsart University (Seoul, Korea), 2019/09/24 (Talk).


We recently identified that iSN04, an 18-nt oligodeoxynucleotide with telomere sequence, strongly promotes skeletal muscle differentiation. Since iSN04 is incorporated into cytoplasm and works depending on its molecular structure, iSN04 is considered to induce myogenic differentiation by interacting with its target protein(s) within the cells. Protein precipitation and mass spectrometry analysis identified that nucleolin is one of the iSN04-binding proteins. Nucleolin is known as a shuttle protein on cell surface to transport nucleotides into cytoplasm or nucleus. Although oligodeoxynucleotides are generally recognized by Toll-like receptors (TLRs), our data suggested that iSN04 is uptaken by nucleolin involved in lipid raft.

Next, we investigated the iSN04-dependent gene expression by RNA sequencing (RNA-seq). Total RNAs were extracted from the human myoblasts treated with 30 uM of iSN04 for 24 hours, then subjected to RNA-seq. 60,448 RNA products were quantified as the fragments per kilobase per million reads (FPKM). In accordance with the result of iSN04-binding protein, iSN04 did not affect TLR signaling pathway. 899 genes were differentially expressed (> 1.5-fold, p < 0.05) between control and iSN04 groups. We examined their function by ontology analyses. The genes suppressed by iSN04 significantly contained the cell cycle-related clusters. On the other hand, the genes induced by iSN04 formed a lot of myogenic clusters such as muscle contraction and sarcomeric proteins. Furthermore, we found that iSN04 also increased the expression of TGF-β signal-related genes. It suggests that iSN04 acts on TGF-β pathway to suppress cell proliferation and promote myogenic differentiation.

Protein precipitation assays and comprehensive gene expression analyses demonstrated that iSN04 is incorporated by nucleolin but not by TLRs, and modulates gene expression program of myoblasts to change the cell fate toward terminally-differentiated myocytes. Elucidating the action mechanism of iSN04 presents important information for its clinical application for age- and disease-related muscular atrophy.

Keywords: myoblast, oligodeoxynucleotide, RNA sequencing, skeletal muscle.