Abstract
In the present study, the roles of a novel long non-coding RNA (lncRNA), lnc-GD2H, in promoting C2C12 myoblast proliferation and differentiation and muscle regeneration were investigated by quantitative polymerase chain reaction, western blotting, Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine (EdU), immunofluorescence staining, luciferase reporter, mass spectrometry, pulldown, chromatin immunoprecipitation, RNA immunoprecipitation assay, wound healing assays, and cardiotoxin (CTX)-induced muscle injury assays. It was observed that lnc-GD2H promoted myoblast proliferation as evidenced by the enhancement of the proliferation markers c-Myc, CDK2, CDK4, and CDK6, percentage of EdU-positive cells, and rate of cell survival during C2C12 myoblast proliferation. Additional experiments confirmed that c-Myc bound to the lnc-GD2H promoter and regulated its transcription. lnc-GD2H promoted cell differentiation with enhanced MyHC immunostaining as well as increased expression of the myogenic marker genes myogenin (Myog), Mef2a, and Mef2c during myoblast differentiation. Additional assays indicated that lnc-GD2H interacted with NACA which plays a role of transcriptional regulation in myoblast differentiation, and the enrichment of NACA at the Myog promoter was impaired by lnc-GD2H. Furthermore, inhibition of lnc-GD2H impaired muscle regeneration after CTX-induced injury in mice. lnc-GD2H facilitated the expression of proliferating marker genes and formed a feedback loop with c-Myc during myoblast proliferation. In differentiating myoblasts, lnc-GD2H interacted with NACA to relieve the inhibitory effect of NACA on Myog, facilitating Myog expression to promote differentiation. The results provide evidence for the role of lncRNAs in muscle regeneration and are useful for developing novel therapeutic targets for muscle disorders.
Highlights
Skeletal muscle accounts for more than 40% of the human body weight and stores approximately 50–75% of human protein in various forms as the largest protein pool (Frontera and Ochala, 2015)
Expression level of lnc-GD2H gradually increased during proliferation and differentiation (Figure 1B). c-Myc increased during proliferation, but it decreased after the beginning of differentiation (Figure 1C)
The results indicated that lnc-GD2H may be a myogenic factor in proliferation and differentiation
Summary
Skeletal muscle accounts for more than 40% of the human body weight and stores approximately 50–75% of human protein in various forms as the largest protein pool (Frontera and Ochala, 2015). Skeletal muscle development/regeneration involves the activation of muscle stem cells, as well as proliferation, differentiation, and muscle fiber fusion of myoblasts (Silva et al, 2019). Myogenic regulatory factors (MRFs) include myoblast determination protein (MyoD), myogenic factor 5 (Myf5), myogenin (Myog), and muscle-specific regulatory factor 4 (MRF4), which mainly regulate the proliferation, differentiation, and fusion of myoblasts. The myocyte enhancer factor 2 (Mef2) family consists of Mef2a, Mef2b, Mef2c, and Mef2d, which regulate the expression of MRFs and modulate the skeletal muscle-specific transcription process involving MRFs (Liu et al, 2020). Among the above-mentioned muscle-specific factors, Myog is a key transcription factor in skeletal muscle growth, development, and regeneration (Hasty et al, 1993; Zhang et al, 2018a). Homozygous Myog-mutant mice survived fetal development but died immediately after birth and exhibited a severe reduction in all skeletal muscle (Hasty et al, 1993)
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