Downregulated miR-204 Promotes Skeletal Muscle Regeneration

Skeletal muscle is the most abundant and a highly plastic tissue of the mammals, especially when it comes to regenerate after trauma, but there is limited information about the mechanism of muscle repair and its regeneration. In the present study, we found that miR-204 is downregulated after skeletal muscle injury. In vitro experiments showed that over-expression of miR-204 by transfecting with miR-204 mimics suppressed C2C12 cell proliferation, migration, and blocked subsequent differentiation, whereas inhibition of miR-204 by transfecting with miR-204 inhibitor showed the converse effects. Furthermore, through the dual luciferase reporter system, we demonstrated that miR-204 can target the 3'UTR regions of Pax7, IGF1, and Mef2c and inhibit their expression. Taken together, our results suggest that Pax7, IGF1, and Mef2c are the target genes of miR-204 in the process of myoblasts proliferation, cell migration, and differentiation, respectively, and may contribute to mouse skeletal muscle regeneration. Our results may provide new ideas and references for the skeletal muscle study and may also provide therapeutic strategies of skeletal muscle injury.


Introduction
Tissue and organ regeneration is common in metazoans, and it is puzzling that regenerative capacity has evolved losses in higher animals [1]. Skeletal muscle is one of the mammalian tissues with a strong regenerative capacity [2]. Skeletal muscle represents about 30~40% of the total body mass in animals [3]. It is not only a motor organ but also an important energy metabolism and endocrine organ. The ability of the skeletal muscle to regenerate depends on the muscle satellite cells, which are located between the sarcolemma and the basal layer of the skeletal muscle fibers [4]. Usually, the muscle satellite cells remain static, can be activated by being injured, and then change from a static state to a myogenic process. Some of the muscle satellite cells that have proliferated by asymmetric division migrate to the damaged part to differentiate into the damaged part of the myotube repair, and the other part returns to the quiescent state, refilling the satellite cell pool [5].
The activation [6], proliferation [7], migration [8], and differentiation [9] of satellite cells are the main processes of muscle regeneration, and a large number of genes have been found to be involved in the process of skeletal muscle regeneration [10]. C2C12 myoblasts, a cell line that is derived from the satellite cells isolated from murine skeletal muscle, are the best immortalized model to study the reserved myoblast function of satellite cells [11,12]. miRNAs are a class of noncoding RNAs of about 22 nt in length that function by inhibiting their target gene expression. With the deepening of miRNA research, some miRNAs have also been found to be involved in the regulation of skeletal muscle regeneration [13]. In recent years, miR-204 has been found to be associated with many biological processes, such as cancer [14], inflammation [15], and lipid metabolism [16], but the role of miR-204 in skeletal muscle regeneration is not fully understood.
In this study, we constructed an acute muscle injury model of skeletal muscle in mice, using cardiotoxin (CTX) to detect the expression level of miR-204 during muscle regeneration. Additionally, C2C12 myoblasts were used as an in vitro model to investigate the function of miR-204 in the proliferation, migration, and differentiation of C2C12 myoblasts by overexpression or inhibition of miR-204. All the results of this study may be able to elucidate the effects of miR-204 on skeletal muscle regeneration and provide valuable information for the diagnosis and treatment of muscle damage. 2.2. Tissue Section. The obtained mouse TA was fixated with 4% paraformaldehyde; then, the tissues were dehydrated using ethanol, infiltrated, and embedded using paraffin; and finally, the tissue sections (4-5 μM thickness) were stained with hematoxylin and eosin (H&E) for histological analysis.

Cell Proliferation
Assay. Cell proliferation assays refer to previous studies [19]. The CCK-8 kit (Beyotime, Shanghai, China) was used at 24 hours, 48 hours, and 96 hours after transfection according to the manufacturer's protocol. Forty-eight hours after transfection, 10μM EdU (Ribobio, Guizhou, China) was added to the growth medium, and the cells were cultured for 2 h. Subsequently, the EdU staining was performed according to the manufacturer's protocol.

Scratch
Test. The C2C12 myoblasts were manually scraped off with a p200 pipette tip to create scratches [20,21]. After the scratch defect was made, the cells were washed in PBS and cultured in DMEM. The C2C12 myoblasts were then incubated at 37°C for 0, 12, or 24 hours; observed; and photographed.

Quantitative
Real-Time PCR (qRT-PCR). qRT-PCR was performed using the SYBR® Premix Ex Taq™ kit (TaKaRa) on a CFX96 Real-Time PCR detection system (Bio-Rad, Richmond, CA, USA). The relative expression of mRNAs and miRNAs was calculated using the 2 −ΔΔCt method [24]. The sequences of primers for qRT-PCR are listed in Table 1. 2.9. Statistical Analyses. All quantitative results are presented as mean ± SD. Statistical analyses were conducted using the SPSS 20.0 software. Unpaired Student's t-test was performed for comparison between the treated and untreated control (NC) groups, and one-way ANOVA analysis was conducted for comparing multiple groups followed by a Dunnett's method post hoc analysis. Statistical significance was defined as p < 0:05.

Downregulation of miR-204 Expression after Muscle
Injury. After CTX (cardiotoxin) injection, a mouse TA (tibialis anterior) muscle injury model was successfully constructed. The HE staining of the TA muscle showed that a large number of muscle fibers were dissolved after CTX injection for 6h, and inflammatory infiltration occurred after 1d. A large number of macrophages appeared at 3 days, and new muscle fibers were formed at 7 days. On 21d, the muscle fibers were basically repaired, but there were still a certain number of central nuclear muscle fibers (Figure 1(a)). In the process of muscle regeneration, the expression level of 2 BioMed Research International Pax7, a marker of muscle satellite cells, showed a pattern of increasing first and then decreasing (Figure 1(b)). Compared with the control group, the expression level of miR-204 showed a trend of decreasing first and then increasing and maintained a lower state throughout the muscle regeneration process (Figure 1(c)).

Pax7 Is a Target Gene of miR-204 That Regulates the Proliferation of C2C12 Myoblasts
To further investigate the function of miR-204 during skeletal muscle regeneration, the TargetScan software and sequence alignment analysis were applied in the miR-204 target finding. Then, we found miR-204 binding sites in the 3'UTR region of Pax7 (Figure 2(a)). Subsequently, we used the dual-luciferase reporter system and found that the overexpression of miR-204 significantly inhibited the luciferase activity of the wild type of Pax7 (Figure 2(b)). As shown in Figure 2(c), compared with the control group, the expression of miR-204 in the mimic group was elevated by approximately 5-fold. However, the expression was down to 31% in the inhibitor group. Compared with the negative control group, the expression of Pax7, CCNB, CCND, CCNE, and CDK4 in C2C12 myoblasts was significantly downregulated by the miR-204 mimic. Meanwhile, the miR-204 inhibitor significantly promoted the expression of these genes (Figures 2(c) and 2(e)). Furthermore, the CCK8 results confirmed that miR-204 suppressed C2C12 myoblast proliferation (Figure 2(f)). In addition, these results were further verified by the EdU assay, that miR-204 mimic decreased the ratio of EdU-positive cells (Figures 2(g) and 2(h)). All these results suggest that miR-204 can inhibit C2C12 myoblast proliferation probably by targeting Pax7.
3.2. IGF1 Is a Target Gene of miR-204 That Regulates the Migration of C2C12 Myoblasts. The migration of satellite cells to the injury site is a key process in the regeneration of skeletal muscles [25]. IGF1 was previously reported to involve in cell migration, as well as in skeletal muscle hypertrophy [26,27]. Meanwhile, miR-204 has also been found to inhibit the migration of osteosarcoma [28]. Taken together, it is possible; therefore, we hypothesized that IGF1 and miR-204 are related to the myocyte migration. Interestingly, we found that the 3'UTR region of the IGF1 has binding sites

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for miR-204 (Figure 3(a)). Further, we confirmed the binding of miR-204 to IGF1 by a dual-luciferase reporter system (Figure 3(b)). The miR-204 mimic significantly inhibited the expression of IGF1 and Pax3, which is the muscle satellite cell migration marker gene (Figure 3(c)). In addition, the scratch test showed that the miR-204 mimic significantly inhibited cell migration, while the miR-204 inhibitor significantly promoted cell migration (Figures 3(d) and 3(e)). All these results suggest that miR-204 may inhibit the migration of C2C12 myoblasts through IGF1.

Mef2c Is a Target Gene of miR-204 That
Regulates the Differentiation of C2C12 Myoblasts. Further, we found and confirmed the binding relationship between miR-204 and Mef2c (Figures 4(a) and 4(b)), which was reported to promote myoblasts differentiation. Compared with the negative control group, the expression level of miR-204 in the mimic group was increased by approximately 21-fold in C2C12 myotubes. However, the expression of miR-204 was down to 45% in the inhibitor group (Figure 4(c)). Figure 4(d), compared with the control group, the expression of MyoG, Mef2c, and MyHC in C2C12 myoblasts was significantly downregulated by the miR-204 mimic. On the contrary, the expression of MyoD, Mef2c, and MyHC was significantly increased after the miR-204 inhibitor transfection (Figure 4(d)). MyHC is a myogenic differentiation marker of myocytes, which was stained by MyHC antibody. The immunocytochemistry results showed that myocytes fusion rate was significantly decreased after the transfection of miR-204 mimic, whereas the fusion index was increased by transfection of miR-204 inhibitor (Figures 4(e) and 4(f)).

Discussion
In the present study, we found that miR-204 is downregulated during skeletal muscle regeneration in vivo. The dualluciferase reporter system and qRT-PCR demonstrated that miR-204 can bind to the 3'UTR of Pax7, IGF1, and Mef2c and then inhibit their expression level. Cell proliferation assay, scratch assay, and differentiation assay showed that  BioMed Research International miR-204 can inhibit the proliferation, migration, and differentiation through its corresponding targets in C2C12 myoblasts, respectively ( Figure 5).
Pax7 is exclusively expressed in satellite cells and plays a crucial role in self-renewal and myogenic potential maintenance in adult skeletal muscle [29]. In the present (f) Cell counts measured using the Cell Count Kit 8 (CCK-8) method. n = 6. (g, h) The EdU assay was carried out after a 48h transfection. C2C12 myoblasts undergoing DNA replication were stained by EdU (red), and cell nuclei were stained with Hoechst (blue). Scale bar = 50 μm, n = 3. Data are presented as means ± SD. * p < 0:05, * * p < 0:01. 5 BioMed Research International study, it was found that the expression level of Pax7 increased significantly on the 1st day after muscle injury, reached the highest level at 3d, and began to fall back after 7d. miR-204 exhibits an opposite expression pattern to Pax7. All these results suggest that miR-204 may also play a crucial role in skeletal muscle regeneration. Furthermore, the dual-luciferase reporter system confirmed the binding relationship between miR-204 and Pax7. In previous researches, miR-204 has been reported to have the ability to inhibit the proliferation of a variety of normal cells [30] and cancer cells [31]. In our study, we consistently found that miR-204 can inhibit the expression of CCNB, CCND, CCNE, and CDK4, which promote cell cycle progression [32,33]. In addition, CCK8 and EdU assays showed that miR-204 mimic significantly inhibited cell proliferation while the miR-204 inhibitor significantly promoted cell proliferation.
Pax3 and IGF1 were found to be involved in cell migration [34,35]. miR-204 has also been found to inhibit the migration of osteosarcoma [28]. Interestingly, we found and confirmed the binding relationship between miR-204 and IGF1. In this study, cell scratch assays also showed that miR-204 can inhibit the migration of C2C12 myoblasts. These results suggest that the role of miR-204 in inhibiting cell migration may exist among various types of cells.
When the satellite cells complete proliferation and migrate to the muscle injury site, the myocytes begin to   [2,36]. MRFs (myogenic regulatory factors) and Mef2c (myocyte enhancer factor 2c) have the ability to promote satellite cell differentiation, which were essential during the myogenesis process [22,27]. During the differentiation of C2C12 myoblasts, miR-204 inhibited its target gene Mef2c and also decreased the expression level of MyoD, MyoG, and MyHC. And further immunofluorescence staining verified that miR-204 inhibited the myocyte differentiation through Mef2c.

Conclusion
In summary, we focused on the expression and function of miR-204 during skeletal muscle regeneration in the present study. The results suggest that the downregulation of miR-204 expression may be associated with skeletal muscle regeneration. Furthermore, our results show that inhibition of miR-204 promoted the expression of Pax7, IGF1, and Mef2c, which are the target genes of miR-204, and are also closely related to skeletal muscle regeneration. These results suggest that miR-204 may regulate the activation, proliferation, migration, and differentiation of myocytes through Pax7, IGF1, and Mef2c. Our findings reveal the potential of miR-204 in the treatment of skeletal muscle-related diseases.

Data Availability
The data used to support the findings of this study are included in the article.