miR-145a-5p Promotes Myoblast Differentiation

MicroRNAs are a class of 18–22-nucleotide noncoding RNAs that posttranscriptionally regulate gene expression and have been shown to play an important role during myoblast differentiation. In this study, we found that the expression of miR-145a-5p was gradually increased during C2C12 myoblast differentiation, and miR-145a-5p inhibitors or mimics significantly suppressed or promoted the relative expression of specific myogenesis related marker genes. Moreover, overexpression or inhibition of miR-145a-5p enhanced or repressed the expression of some special genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, including Wnt5a, LRP5, Axin2, and β-catenin. These results indicated that miR-145a-5p might be considered as a new myogenic differentiation-associated microRNA that can promote C2C12 myoblast differentiation by enhancing genes related to myoblasts differentiation.


Introduction
Skeletal muscle is the major storage tissue for postprandial glucose. Indeed, approximately 75% of insulin-dependent glucose in the plasma is stored in the skeletal muscle [1]. In addition, as a major site of metabolic activity, the skeletal muscle is a remarkably complex organ constituted by many different cell types, playing important functional roles in injury, disease, and regeneration [2,3]. Therefore, to explore the mechanism of muscle development may contribute to a better understanding of these diseases and muscle development. Previous studies showed that some transcription factors could coordinate the expression of genes involved in muscle growth, morphogenesis, differentiation, and contractility, such as MyoD, MyoG, myf5, MRF4, ACTA1, and MyHC [4,5]. In addition, skeletal muscle development involves several signaling pathways. For instance, the Wnt signaling pathway that involves members of the Wnt family initiates the myogenic differentiation in the epaxial and hypaxial musculature [6,7]. MicroRNAs are a class of approximately 18−22-nucleotide, single-stranded, noncoding small RNA molecules that posttranscriptionally regulate gene expression and play important roles in cell embryogenesis, cell differentiation, carcinogenesis, tumorigenesis, and apoptosis [8,9]. Recently, the involvement of miRNAs in the regulation of skeletal muscle differentiation has been demonstrated [10,11], revealing that miR-145 regulates smooth muscle fate and plasticity, taking part in diverse biological processes through Wnt signaling pathway [12,13]. However, little is known about whether miR-145a-5p plays a role in the process of myogenic differentiation. Moreover, a connection between miR-145a-5p and Wnt signaling pathway during myogenic differentiation has not been yet fully demonstrated and described.
C2C12 myoblast cells are a common model to study muscle differentiation [14][15][16][17]. In the present study, we showed that the expression level of miR-145a-5p was gradually increased during C2C12 myoblast differentiation, and reducing or increasing miR-145a-5p significantly suppressed or F: CTCGCTTCGGCAGCACA 58 R: AACGCTTCACGAATTTGCGT enhanced the relative expression levels of marker genes, when transfecting miR-145a-5p inhibitors or mimics into C2C12 cells. Additionally, the transfection of miR-145a-5p inhibitors or mimics repressed or increased the expression of genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, when compared to negative control. These data indicated that miR-145a-5p might be characterized as a new myogenic differentiation-associated microRNA and can promote C2C12 myoblast differentiation by enhancing genes related to myoblast differentiation.

Cell
Culture. C2C12 myoblasts (Stem Cell Bank, Chinese Academy of Sciences) were maintained at 37 ∘ C and 5% CO 2 in growth medium containing Dulbecco's modified Eagle's medium (DMEM, Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum before being induced to differentiate. When cells reached 80% confluence, they were digested with 0.25% trypsin and then seeded in 12-well plates. When cell density reached 70-80%, the medium was switched to differentiation medium containing DMEM and 2% horse serum (Gibco).
the expression levels of individual mRNAs, while microRNA expression was normalized against the expression of U6.

Immunocytochemical Analysis.
After transfection and induced myogenic differentiation, C2C12 myoblasts cultured in 12-well plates were washed three times with phosphatebuffered saline (PBS) and fixed in 4% paraformaldehyde for 15 min. After further PBS washes (and for each step thereafter), cells were then permeabilized with 0.5% Triton X-100 prior to blocking in 2% goat serum (diluted in PBS). After blocking, cells were incubated with an anti-myosin primary antibody at 37 ∘ C for 2 h and then fluorescent secondary antibodies at 37 ∘ C for 1 h. The nuclei were stained with Hoechst (Boster, Wuhan, China) for 10 min. Images were captured using a Nikon TE2000 microscope (Nikon, Tokyo, Japan).

Statistical Analysis.
All data are presented as means ± standard error of the mean (SEM). SPSS 22.0 software was used for statistical analysis. Comparisons were made using the one-way analysis for the least three parametric groups, and Student's -test was used for two parametric groups. A value of < 0.05 indicated a significant difference.   To explore the potential role of miR-145a-5p in myoblast differentiation, we analyzed the temporal expression pattern of miR-145a-5p during C2C12 myoblast differentiation. As shown in Figure 1(b), as compared to zero day of the differentiation, the expression levels of miR-145a-5p in C2C12 were increased more than 25 times during differentiation ( < 0.01). This result showed a similar fluctuation to what was observed in miR-148a [18] and miR-486 [19]; both of them were upregulated during myoblast differentiation and played important roles in the process. Therefore, the increased expression of miR-145-5p could be also associated with miR-145a-5p important role in C2C12 cells myoblast differentiation.
As compared with the control group, miR-145a-5p inhibitors caused a significantly progressive decrease in relative mRNA levels of MyoD and MyoG. By contrast, transfection of miR-145a-5p mimics markedly increased their expression levels compared to the negative control. Previous results showed that MyoD and MyoG are master myogenic transcriptional regulatory factors that activate a number of muscle-specific genes to drive muscle cell differentiation [20,21]. To further investigate the role of miR-145a-5p in myogenic differentiation, we performed the immunofluorescence of MyoD and MyoG (Figure 2(d)). Similarly, myotubes were significantly decreased or increased in the cells expressing the miR-145a-5p inhibitors or mimics as compared to the control group on the 8th day after transfection.
To better understand the mechanism of miR-145a-5p during myoblasts differentiation, we analyze the expression levels of more marker genes related to myoblasts differentiation. As shown in Figure 3, the expression levels of genes related to myoblasts differentiation were downregulated during myoblast differentiation, when miR-145a-5p was inhibited in C2C12 cells. However, expression levels of those maker genes were markedly increased in cells transfected with miR-145a-5p mimics, when compared to the control group. myf5, MyoD, MyoG, MRF4, ACTA1, and MyHC are key regulation factors during myoblasts differentiation [4,22]. Previous studies showed that miR-181 indirectly upregulated the expression of MyoD to enhance myoblasts differentiation, while miR-139 inhibited myoblasts differentiation by repressing the expression of MyoG and MyHC [23,24]. In the current study, almost all results supported our hypothesis that miR-145a-5p may promote the C2C12 myoblasts differentiation by enhancing some special genes related to myoblasts differentiation.

The Effect of miR-145a-5p on Wnt Pathway.
Previous studies showed that miR-145 takes part in biological processes by regulating Wnt signaling in cancer [12,13]. However, a potential connection between miR-145a-5p and Wnt signaling pathway during myogenic differentiation is not yet clearly demonstrated. As shown in Figure 4(a), we analyzed the expression of the main upstream factors of the canonical Wnt signaling pathway at the different time points before and after C2C12 cell differentiation. The mRNA levels of Wnt5a, LRP5, Axin2, and -catenin gradually increased with the increased C2C12 differentiation. In addition, as expected, inhibiting or enhancing miR-145a-5p resulted in a significant decrease or increase in the expression levels of Wnt5a, LRP5, Axin2, and -catenin (Figures 5 and 6). Skeletal muscle development is a complex process, involving several signaling pathways. Some previous studies indicated that Wnt signaling regulates myogenic differentiation in the development of avian wings [25], and it is involved in the switching from cell proliferation to myogenic differentiation of mouse myoblast cells [26]. In particular, Wnt5a can activate both myf5 and MyoD [27]. In the present study, miR-145a-5p inhibitors or mimics significantly inhibited or enhanced the expression of MyHC and myf5 (Figures 4(b) and 4(c)) and the main upstream factors of the canonical Wnt signaling pathway during myoblast differentiation. This result indicated that miR-145a-5p may promote the C2C12 myoblasts differentiation and possessed a connection with Wnt signaling pathway during myogenic differentiation.

Conclusions
In the present study, we identified miR-145a-5p as a new myoblast differentiation-associated miRNA in C2C12 cells. miR-145a-5p not only possessed a positive effect on C2C12 myoblast differentiation but also promoted the expression of genes (Wnt5a, LRP5, Axin2, and -catenin) involved in the endogenous Wnt signaling during C2C12 myoblast differentiation. These results suggested that miR-145a-5p may promote the expression of specific myoblast differentiation markers and myotube differentiation. From a clinical point of view, miR-145a-5p could also be considered as a novel disease marker for the diagnosis of muscle wasting or for monitoring its progression, leading to useful therapies for this type of disease.