LncRNA XLOC_015548 affects the proliferation and differentiation of myoblasts via the MAPK signaling pathway

Abstract

In recent years, an increasing number of studies have reported that long non-coding RNAs (lncRNAs) play essential regulatory roles in myogenic differentiation. In this study, a specific LncRNA XLOC_015548 (Lnc000280) was identified. However, little research has explored its mechanism of action by constructing XLOC_015548 gene editing cell models. In this study, relevant sequences were obtained according to the RNA-seq results. Subsequently, XLOC_015548 knockdown and over-expression lentiviral vectors were constructed, and the C2C12 myoblast cell line was transfected to prepare the XLOC_015548 gene-edited myoblast model. The in vitro analysis revealed that over-expression of XLOC_015548 significantly promoted the proliferation and differentiation of myoblasts and the formation of myotubes, whereas the opposite result was obtained in the knockdown group. XLOC_015548 regulated myogenic differentiation and affected the expression of myogenic differentiation regulators such as Myod, myogenin, and MyHC. Regarding the signaling pathway, we found that XLOC_015548 correlated with the phosphorylation level of MAPK/MEK/ERK pathway proteins. And the degree of phosphorylation was positively correlated with the protein expression of myogenic differentiation regulators. In conclusion, a new gene-edited myoblast model was constructed based on the lncRNA regulator XLOC_015548. The in vitro cell experiments verified that XLOC_015548 had regulatory effects on muscle growth and myoblast differentiation. These findings provide a laboratory foundation for the clinical application of lncRNAs as regulatory factors in the treatment of disuse muscle atrophy.

Keywords: Long non-coding RNA XLOC_015548; MAPK signaling pathway; RNA sequencing; muscle; myoblast differentiation; myogenesis.

Figure 1.

Construction of the XLOC_015548 gene editing cell model and the effect of XLOC_015548 on cell viability and proliferation. (A) After lentiviral transfection, the GFP level of the cells in each group was observed using a fluorescence microscope. (B, C) The mRNA expression of XLOC_015548 in each group was determined using RT-qPCR. (D, E) The EdU assay was performed to identify the survival rate and proliferation ability of the cells in each group after lentiviral transfection. (F) CCK-8 was used to detect the growth rates of C2C12 cells. Each experiment was repeated three times, and the data were expressed as mean ± standard deviation. Scale bars, 100 μm. *P < 0.05; **P < 0.01.

Figure 2.

Effects of XLOC_015548 on myotube activity and contractility. (A) The differentiation medium promoted the fusion of cells in each group to form myotubes. (B–D) The immunofluorescence assay was used to detect the positive rate of MyHC in the myotubes after differentiation in each group. (E–G) The myotube contraction assay was performed to detect the contractility and activity of the myotubes. Each experiment was repeated three times, and the data were expressed as mean ± standard deviation. Scale bars, 100 μm. *P < 0.05; **P < 0.01.

Figure 3.

XLOC_015548 promoted the differentiation of myoblasts. (A, G) RT-qPCR was performed to determine the mRNA expression of Myod in each group. (B–D and H–I) Western blot analysis was conducted to identify the protein expression of Myod and MEK/ERK signaling pathway in lentivirus infection groups. Each experiment was repeated three times, and the data were expressed as mean ± standard deviation. *P < 0.05; **P < 0.01.

Figure 4.

XLOC_015548 activated the MAPK/MEK/ERK signaling pathway and promoted myogenic differentiation. (A–F) The expression of phosphorylated proteins of MEK1/2 and ERK1/2 in the MAPK pathway and myoblast-related proteins were detected using western blot analysis in XLOC_015548-siRNA groups. (G–L) After treatment with 1 μM U0126, the expression level of phosphorylated proteins of MEK/ERK or myogenic differentiation factors was detected using western blot analysis. Each experiment was repeated three times, and the data were expressed as mean ± standard deviation. *P < 0.05; **P < 0.01.