MicroRNA‑23a‑5p mediates the proliferation and differentiation of C2C12 myoblasts.

Skeletal myogenesis is a highly ordered and complex biological process that is mediated by numerous regulatory factors. In previous studies, it has been demonstrated that microRNAs (miRs) and long non‑coding RNAs (lncRNAs) serve key roles in skeletal myogenesis. The present study showed that the expression levels of miR‑23a‑5p showed a dynamic change from decrease to increase during C2C12 myoblast proliferation and differentiation. Functional analysis using 5‑ethynyl‑2'‑deoxyuridine proliferation and Cell Counting Kit‑8 detection assays indicated that overexpression of miR‑23a‑5p significantly promoted C2C12 myoblast proliferation compared with the negative control. In addition, in C2C12 myoblasts transfected with miR‑23a‑5p mimics, increased expression levels of regulators associated with cell proliferation (Cyclin E, CCND1 and Cyclin B) were observed compared with the negative control. By contrast, overexpression of miR‑23a‑5p decreased the expression levels of specific‑myogenesis factors (MyoD, MyoG and Myf5) and decreased C2C12 myoblast differentiation. Luciferase activity assays indicated that miR‑23a‑5p suppressed the luciferase activity of lncDum. Further analysis demonstrated that miR‑23a‑5p not only showed an opposite expression level pattern compared with lncDum, which was first increased and then decreased, but also had an opposite effect on the proliferation and differentiation of C2C12 myoblasts compared with lncDum which inhibited cell proliferation and promoted cell differentiation. Taken together, these results indicated that miR‑23a‑5p may mediate the proliferation and differentiation of C2C12 myoblasts, which may be involved in lncDum regulation.

Introduction

The basic functions of the skeletal musculature include facilitating locomotor activity, postural behavior and breathing (1). In addition, the skeletal musculature is a key metabolic organ in the human body, which is involved in regulating metabolite homeostasis and maintaining metabolic health via processes such as mediating glucose uptake and insulin sensitivity (2). Skeletal muscle has been identified as a secretory organ, which may exert autocrine, paracrine or endocrine effects to affect the function of adipose tissue or the cardiovascular system (3). Therefore, impaired or loss of function of skeletal muscle can affect functional capacity and increase the risk of a number of diseases, such as diabetes mellitus (4), muscle atrophy (5) and cancer (6). Skeletal myogenesis is a complex biological process that involves myoblast proliferation, migration and differentiation, as well as fusion of multicellular myotubes into contractile skeletal muscle fibers (7). In addition, according to skeletal muscle contractility and expression levels of myosin heavy chain (MyHC) subtypes, skeletal muscle fibers are classified into two major types: Fast- (MyHC2b and MyHC2×) and slow-twitch fibers (MyHC1 and MyHC2a) (8–10). A number of transcription factors mediate skeletal myogenesis (11–15), including the myogenic regulatory family (MRF) and the myocyte enhancer 2 family (MEF2), which are among the most studied gene families involved in regulating skeletal myogenesis (14). The MRF family, also known as the MyoD family, consists of MyoD, MyoG, Myf5 and MRF4 (14), and serves a key role in regulating specific gene transcription in muscle cells, as well as cell growth cycle and differentiation (16). For instance, MyoD and Myf5 induce expression levels of myogenin and other MEF2 family transcription factors (17). Subsequently, myogenin and MEF2 family members cooperate in the activation of muscle structural genes during differentiation and the formation of multinucleated myotubes (17). Megeney et al (18) demonstrated that mice lacking both MyoD and dystrophin displayed a marked increase in severity of myopathy leading to premature death, suggesting a role for MyoD in muscle regeneration.

Evidence has demonstrated that microRNAs (miRNAs/miRs) are a class of non-coding RNAs 17–24 nucleotides in length (19,20), which serve key roles in numerous physiological and pathological processes, such as cell proliferation, cell differentiation, or tumorigenesis (21–23). A number of studies have shown that miRNAs regulate the expression levels of key functional genes and transcription factors at the posttranscriptional level, and induce mRNA degradation or translation inhibition by interacting with the 3′ untranslated regions (UTRs) of their target mRNAs (20,24). Involvement of miRNAs in skeletal muscle development and function has previously been reported (25,26). A number of studies have demonstrated that miRNAs function as key regulators of myogenesis via regulating myoblast proliferation and differentiation (25,27,28). miR-1, for example, promotes myogenesis via targeting histone deacetylase 4, a transcriptional repressor of the myogenesis gene (29). miRNA-133 enhances myoblast proliferation by repressing serum response factor (29). miRNA-206 regulates skeletal muscle satellite cell proliferation and differentiation by repressing Pax7 (26), and miRNA-139-5p regulates C2C12 cell myogenesis by blocking the Wnt/β-catenin signaling pathway (30). In addition, miRNA-27b regulates Pax3 protein levels, and downregulation of Pax3 causes rapid entry into the myogenic differentiation program (31). Changes in miRNA expression levels have been reported to be associated with skeletal myogenesis (32,33).

Long non-coding RNAs (lncRNAs), a class of non-protein-coding RNAs >200 nucleotides in length, have been identified as competing endogenous RNAs (ceRNAs) that sponge miRNAs via complementary base pairing to regulate biological process (34). To date, numerous lncRNAs have been identified in mice and humans, among which some serve important roles in muscle development and myogenesis (35,36). A number of studies have indicated that expression levels of miR-23a and miR-23b are increased in the early stage of C2C12 differentiation, and that these miRNAs are involved in the regulation of TrxR1 expression levels via direct binding to the 3′ UTR of TrxR1 mRNA (37,38). Wang et al (10) reported that miR-23a-3p downregulates the expression levels of Myh1, 2 and 4 via targeting the 3′ UTR of mRNAs, thereby inhibiting myogenesis of C2C12 myoblasts and fast-twitch MyHC isoforms. However, the role of miR-23a-5p in myoblast proliferation and differentiation has not yet been fully elucidated. The present study demonstrated that miR-23a-5p affected myoblast proliferation and differentiation. It was hypothesized that miR-23a-5p promoted C2C12 myoblast proliferation and inhibited differentiation, which may be involved in lncDum regulation.

Materials and methods

Animal experiments

A total of 21 female C57BL/6 mice (Dashuo Co., Ltd.) had free access to food and water in natural light cycle and 37°C temperature conditions. The muscle tissues of 2–6-week-old C57BL/6 mice were collected for RNA isolation. A total of three mice were included each group (2-week-old, 3-week-old, 4-week-old, 5-week-old, 6-week-old C57B/L6 mice). Tissues (liver, spleen, lung, heart, kidney, muscle, inguinal fat, perirenal fat, brain, shoulder fat and gonads fat) of 6-week-old C57BL/6 mice were collected for RNA isolation. In order to establish a muscle atrophy model, muscle atrophy was induced by Dexamethasone (Dex; Beijing Solarbio Science & Technology Co., Ltd.)-mediated injury as described by Morimoto et al (39). In brief, three 10-week-old mice were intramuscularly injected in the left hind leg with 100 µl 10 µM Dex. As a control, the right legs of the same mice were intramuscularly injected with PBS. The injection was performed every 7 days to ensure success. At 14 days after the first injection, muscle samples were collected for RNA isolation.

All animal procedures were approved by the Animal Care and Ethics Committee of Sichuan Agricultural University [approval no. DKY-(S20176903)] and performed in accordance with the institutional guidelines for the care and use of laboratory animals.

Cell culture

C2C12 myoblasts and HeLa cells (Beijing Stem Cell Bank; Chinese Academy of Science) were cultured in growth medium (GM) containing DMEM and 10% FBS (both Gibco; Thermo Fisher Scientific, Inc.). When C2C12 myoblasts reached 80% confluence, GM was replaced with differentiation medium (DM) containing DMEM supplemented with 2% horse serum (Gibco; Thermo Fisher Scientific, Inc.). Cells were cultured at 37°C with 5% CO2.

Cell transfection

miR-23a-5p mimics (cat. no. miR10017019-1-5), negative control (NC) mimics (cat. no. miR01201-1-5) and small interfering (si)RNA molecules directed against lncDum (si-lncDum) were designed and synthesized by Guangzhou RiboBio Co., Ltd. Overexpression of lncDum (p-lncDum) was achieved by cloning lncDum complementary DNA into vector pcDNA3.1 (Beijing Tsingke New Industry TsingKe Biotechnology Co., Ltd.). Briefly, 5×105 or 4×104 C2C12 myoblasts were seeded in 12- or 96-well plates and grown to 80% confluency, then transfected with miRNA-23a-5p mimics, NC mimics, si-lncDum, p-lncDum, empty pcDNA3.1+ vector or siRNA control (si-NC) at a concentration of about 20 µM using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The transfections were carried out every 48 h. Cells were harvested for 6 days, and bright-field microscope was used to observe the ability of C2C12 myoblasts to differentiate into myotubes 6 days post-transfection. The sequences of synthesized RNA oligonucleotides are presented in Table SI.

Reverse transcription-quantitative PCR (RT-qPCR)

RNA from muscle tissue and C2C12 cells was obtained using TRIzol® (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. Reverse transcription was performed using a PrimeScript RT Master Mix kit for mRNA, or a PrimeScript™ miRNA RTPCR kit (both Takara Biotechnology Co., Ltd.) for miRNA, according to the manufacturer's instructions. qPCR was performed using the SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd.) using a CFX96 system (Bio-Rad Laboratories, Inc.). U6 and β-actin served as endogenous controls for miRNA and mRNA, respectively. The expression levels of miRNA and mRNA were quantified using the comparative threshold cycle (2−ΔΔCq) method (40). Primer sequences are presented in Table SII.

Cell proliferation analysis

In brief, C2C12 myoblasts were seeded in 96-well plates, and transfected with miR-23a-5p mimics, NC mimics, si-lncDum, p-lncDum, si-NC or pcDNA3.1+ empty vector, after which cell proliferation was determined at 0, 12, 24, 48, 72 and 96 h using a Cell Counting Kit (CCK)-8 (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. For 5-ethynyl-2′-deoxyuridine (EdU) proliferation analysis, C2C12 myoblasts were treated with 10 µM EdU (Guangzhou RiboBio Co., Ltd.) after 24 h post-transfection and incubated for 3 h at 37°C. EdU staining was performed according to the manufacturer's protocol. Cell nuclei were stained with DAPI (Beyotime Institute of Biotechnology) for 10 min at room temperature. Images (magnification, ×100) of the entire well were captured using an IX53 fluorescent inverted microscope (Olympus Corporation).

Western blotting

Total cellular proteins were extracted using RIPA lysis buffer (Wuhan Servicebio Technology Co., Ltd.) 96 h after transfection. The native protein lysate was collected, and the protein concentration was measured using the BCA protein concentration assay kit (Wuhan Servicebio Technology Co., Ltd.) according to the manufacturer's instructions. Proteins (30–50 µg/per well) were resolved by SDS-PAGE with 10% gels, then transferred to PVDF membranes (EMD Millipore). The membranes were blocked with 5% non-fat milk in 0.5% TBS-Tween-20 (TBST) for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies against β-actin (dilution 1:1,000; cat. no. GB12001, Wuhan Servicebio Technology Co., Ltd.); MyoG (dilution ratio 1:1,000; cat. no. A6664, ABclonal), and MyHC1 (dilution ratio 1:1,000; cat. no. A7564, ABclonal). The membranes were washed with TBST and then incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG; dilution 1:3,000; cat. no. GB23303; Wuhan Servicebio Technology Co., Ltd.) for 30 min at room temperature. The signals were visualized using enhanced chemiluminescent reagent (Wuhan Servicebio Technology Co., Ltd.), and the expression levels of β-actin were used for normalization. The AlphaEaseFC™ software processing system (version 3.2β; Alpha Innotech) was used to analyze the optical density value of the target bands.

Immunofluorescence analysis

For immunofluorescence analysis, C2C12 myoblasts were transfected with miR-23a-5p mimics, NC, si-lncDum, p-lncDum, si-NC or pcDNA3.1+ empty vector during differentiation, then washed three times with PBS, and fixed in 4% paraformaldehyde for 30 min at room temperature. Next, cells were washed three times with PBS, and blocked with 5% normal goat serum in PBS for 1 h at room temperature. Then, cells were incubated with an anti-myosin slow antibody (anti-MyHC1; dilution 1:300; cat. no. bs-9862R; BIOSS) overnight at 4°C, washed three times with PBS, and incubated with a fluorescent secondary antibody (dilution ratio 1:300; cat. no. bs-0295G-FITC; BIOSS) at 37°C for 1 h. Cell nuclei were stained with DAPI (Beyotime Institute of Biotechnology) for 20 min at room temperature. Images (magnification, ×100) were captured using an IX53 fluorescent inverted microscope (Olympus Corporation). The fusion index of myotubes as calculated by Image J software (Image J 1.50i; National Institutes of Health).

Luciferase reporter assay

HeLa cells were seeded in 96-well plates and cultured to 90% confluency prior to transfection. Wild-type (WT-lncDum) and mutant lncDum (Mut-lncDum) were amplified using primers containing XhoI and NotI restriction sites and cloned into a psi-CHECK™-2 promoter vector at the 3′ end of the Renilla gene Beijing Tsingke New Industry TsingKe Biotechnology Co., Ltd.). For the luciferase reporter analysis, 4×104 HeLa cells were seeded in 96-well plates and grown to 80% confluency, then co-transfected with either WT-lncDum or Mut-lncDum in conjunction with either miR-23a-5p mimics or mimics control (Guangzhou RiboBio Co., Ltd.) at a final concentration of 100 nM using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Luciferase activity was measured using a Dual-Glo Luciferase Assay System (Promega Corporation) 24 h after transfection, according to the manufacturer's instructions. Luciferase activity levels were normalized to those of firefly luciferase.

Statistical analysis

Data are presented as the mean ± SEM of three experiments. Statistical analysis was performed using SPSS software (version 22.0; IBM Corp.). Differences between groups were determined using an unpaired Student's t-test. Differences between 3 or more groups were determined by one-way ANOVA, followed by the Least Significant Difference method. P<0.05 was considered to indicate a statistically significant difference.

Results

miR-23a-5p is associated with skeletal myogenesis

miR-23a-5p exhibited a similar expression profile to three myoblast-specific miRNAs [miR-206-3p (41), miR-181a-5p (42) and miR-221 (43); Fig. 1A] in C2C12 myoblasts. The expression levels of miR-23a-5p in numerous types of tissue derived from 6-week-old WT mice were assessed, which demonstrated that miR-23a-5p was highly expressed in muscle, compared with adipose tissue and brain. However, miR-23a-5p was expressed at lower levels in muscle tissue compared with major organs, such as the liver, spleen, lung, heart and kidney (Fig. 1B). Furthermore, miR-23a-5p was highly expressed in the muscle tissue of 2–3-week-old mice, and notably decreased after 3 weeks and remained at low levels as mice aged (Fig. 1C). In order to confirm these results, a muscle atrophy model was established using Dex injection, as previously described (5,44,45). Compared with the control group, Dex treatment caused a significant decrease in miR-23a-5p expression levels in muscle tissue (Fig. 1D). Thus, the present findings indicated that miR-23a-5p may be associated with skeletal myogenesis.

Figure 1.

miR-23a-5p is associated with myogenesis. Reverse transcription-quantitative PCR was used to measure the expression levels of (A) miRs during myoblast proliferation and differentiation, and miR-23a-5p in (B) different types of tissue in 6-week-old mice, (C) muscle tissue of mice of different ages and (D) muscle tissue following treatment with Dex. Data are presented as the mean ± SEM (n=3). **P<0.01. miR, microRNA; Dex, dexamethasone.

miR-23a-5p promotes C2C12 myoblast proliferation

miR-23a-5p exhibited a notably decreased expression level during the proliferation and differentiation of C2C12 myoblasts compared with myogenesis-specific miRNAs, including miR-206-3p, miR-181a-5p, miR-221/222 and miR-29b-3p (Fig. 2A). In order to identify the role of miR-23a-5p in regulating skeletal myogenesis, miR-23a-5p mimics were transfected into C2C12 myoblasts using GM to evaluate whether miR-23a-5p mediates proliferation of these cells. Transfection of miR-23a-5p mimics increased the expression levels of miR-23a-5p by 1,327-fold compared with the NC group, indicating that miR-23a-5p was successfully overexpressed in proliferating C2C12 myoblasts (Fig. 2B). Subsequently, an EdU proliferation assay was performed to evaluate the effect of miR-23a-5p on C2C12 myoblast proliferation; the results demonstrated that miR-23a-5p significantly increased the proliferation of EdU-positive cells compared with NC cells (Fig. 2C and D). These findings were also validated by an CCK-8 assay: Following transfection for 72 h, a greater number of C2C12 myoblasts were observed in the miR-23a-5p mimics group compared with the NC group (Fig. 2E). Furthermore, RT-qPCR analysis demonstrated that the expression levels of Cyclin E and Cyclin B were significantly upregulated in C2C12 myoblasts transfected with miR-23a-5p mimics, compared with the NC group (Fig. 2F). Although increasing miR-23a-5p tended to cause a decrease in P21 expression levels compared with the NC group, the difference was not significant. Taken together, these data indicated that miR-23a-5p positively regulated C2C12 myoblast proliferation.

Figure 2.

miR-23a-5p positively modulates C2C12 myoblast proliferation. RT-qPCR was used to measure expression levels of (A) miRNAs during C2C12 myoblast proliferation and differentiation, and (B) miR-23a-5p in C2C12 myoblasts transfected with miR-23a-5p mimics or NC during proliferation. (C) EdU assay was performed following transfection for 24 h. Cells undergoing DNA replication were stained with EdU (red) and cell nuclei were stained with DAPI (blue). Scale bar, 100 µm. (D) Percentage of EdU-positive cells/DAPI-positive cells was quantified. (E) Cell count was measured by an CCK-8 assay. (F) RT-qPCR analysis of the expression levels of Cyclin E, Cyclin B, CCND1 and P21. Data are presented as the mean ± SEM (n=3). *P<0.05, **P<0.01 vs. NC or as indicated. miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; CCK-8, Cell Counting Kit-8; EdU, 5-ethynyl-2′-deoxyuridine; OD, optical density; NS, not significant.

miR-23a-5p inhibits C2C12 myoblast differentiation

In order to investigate the effect of miR-23a-5p on myoblast differentiation, miR-23a-5p mimics were transfected into C2C12 myoblasts using DM. At 6 days after transfection of miR-23a-5p mimics (Fig. 3A), bright-field microscopy was performed, which demonstrated that miR-23a-5p overexpression markedly inhibited the fusion of myotubes compared with the NC group (Fig. 3B). In order to confirm these results, the expression levels of various myogenic transcription factors, such as MyoD, Myf5, MyoG, MRF4 and MyHC, were evaluated. Transfection with miR-23a-5p mimics resulted in a significant decrease in the expression levels of MyoD, MyoG and Myf5 compared with the NC group (Fig. 3C). Furthermore, MyoG protein expression levels in miR-23a-5p-overexpressing C2C12 cells were assessed. It was revealed that MyoG was downregulated following miR-23a-5p overexpression, as determined via western blotting analysis (Fig. 3D).

Figure 3.

miR-23a-5p negatively modulates C2C12 myoblast differentiation and formation of muscle fiber type. (A) RT-qPCR analysis of the expression levels of miR-23a-5p in C2C12 myoblasts transfected with miR-23a-5p mimics or NC during differentiation. (B) Bright-field microscopy was used to observe the ability of C2C12 myoblasts to differentiate into myotubes at 6 days post-transfection. Scale bar, 100 µm. (C) RT-qPCR analysis of the expression levels of MyoG, MyoD, MRF4, Myf5 and MyHC. (D) Western blotting analysis of MyoG protein levels in C2C12 myoblasts transfected with miR-23a-5p mimics or NC during differentiation. RT-qPCR analysis of (E) expression levels of genes associated with fast- and slow-twitch fibers, and (F) The composition of MyHC (MyHC1, MyHC2a, MyHC2b and MyHC2×) in myoblasts transfected with miR-23a-5p mimics or NC. (G) Western blotting analysis of MyHC1 protein levels in C2C12 myoblasts transfected with miR-23a-5p mimics or NC during differentiation. (H) Immunofluorescence staining of MyHC1 was used to analyze myosin-slow-positive myotubes. The fusion index was calculated as MyHC-positive cells to total nuclei (total DAPI-positive cells). Scale bar, 100 µm. Data are presented as the mean ± SEM of three independent repeats. *P<0.05, **P<0.01. miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; MyHC, myosin heavy chain; NS, not significant.

As muscle fiber type composition is key for muscle biological function and body metabolism, the present study aimed to determine whether miR-23a-5p mediates muscle fiber type composition. RT-qPCR demonstrated that overexpression of miR-23a-5p notably inhibited the expression levels of several genes associated with fast- (Tnni2, Tnnc2, MyHC2b and MyHC2×) and slow-twitch fibers (Tnni1, Tnnc1, MyHC2a and MyHC1) (46) compared with the NC group, indicating that miR-23a-5p may be involved in regulating the composition of skeletal muscle fiber type (Fig. 3E). The expression levels of MyHC1, −2a, −2b and −2×, following transfection of C2C12 myoblasts with miR-23a-5p mimics or NC were further analyzed during differentiation. Overexpression of miR-23a-5p in C212 myotubes decreased the percentage of MyHC1 and increased the percentage of MyHC2a by RT-qPCR (Fig. 3F). In addition, overexpression of miR-23a-5p decreased MyHC1 protein level by western blotting analysis (Fig. 3G), as demonstrated by immunofluorescence analysis of MyHC1 (Fig. 3H). Taken together, these results indicated that miR-23a-5p may affect C2C12 myoblast differentiation and mediate skeletal muscle fiber type composition.

miR-23a-5p regulates C2C12 myoblast proliferation and differentiation by interacting with lncDum

A previous study have demonstrated that lncDum serves a key role in regulating cell differentiation and muscle regeneration (47). In order to confirm the function of lncDum on C2C12 myoblast proliferation and differentiation, si-lncDum, si-NC, p-lncDum or empty pcDNA3.1+ vector were transfected into C2C12 myoblasts during proliferation (Fig. 4A). CCK-8 and EdU assays revealed that inhibition of lncDum significantly promoted C2C12 myoblast proliferation compared with the control (Fig. 4B-D). Consistent with these findings, Cyclin E, CCND1 and Cyclin B were upregulated, and P21 was downregulated in C212 myoblasts that were transfected with si-lncDum (Fig. 4E). Consistent with previous findings (47) that lncDum enhances C2C12 myoblast differentiation, overexpression of lncDum promoted the fusion of myotubes and increased the expression levels of MyoD, MRF4, MyoG, Myf5 and MyHC, whereas lncDum inhibition decreased the fusion of myotubes and downregulated these genes (Fig. 4F-H). In addition, western blotting analysis showed that the expression levels of MyoG were also upregulated by lncDum overexpression and downregulated by lncDum inhibition compared with the control (Fig. 4I), thereby indicating that lncDum promoted myoblast differentiation.

Figure 4.

miR-23a-5p regulates C2C12 myoblast proliferation and differentiation by interacting with lncDum. (A) RT-qPCR analysis of the expression levels of lncDum in myoblasts transfected with si-lncDum, si-NC, p-lncDum or pcDNA3.1+ empty vector during proliferation. (B) EdU assay was performed following 24 h of transfection. Scale bar, 100 µm. (C) Percentage of EdU-positive/DAPI-positive cells was quantified. (D) Cell count was measured via a CCK-8 assay in C2C12 myoblasts transfected with si-lncDum, si-NC, pcDNA3.1+ empty vector or p-lncDum. RT-qPCR analysis of the expression levels of (E) Cyclin E, Cyclin B, CCND1 and P21, and (F) lncDum in myoblasts transfected with si-lncDum, si-NC, pcDNA3.1+ empty vector or p-lncDum during differentiation. (G) Bright-field microscopy was used to observe the ability of C2C12 myoblasts to differentiate into myotubes at 6 days after transfecting with si-lncDum, si-NC, pcDNA3.1+ empty vector or p-lncDum. Scale bar, 100 µm. (H) RT-qPCR measured the expression levels of MyoG, MyoD, MRF4, Myf5 and MyHC. (I) Western blotting analysis of MyoG protein levels in C2C12 myoblasts transfected with si-lncDum, si-NC, p-lncDum or pcDNA3.1+ empty vector during differentiation. (J) lncDum contained a binding site for miR-23a-5p. RT-qPCR analysis of the expression levels of (K) lncDum and miR-23a-5p during myoblast proliferation and differentiation, and (L) lncDum in myoblasts transfected with miR-23a-5p mimics and p-lncDum, NC or p-lncDum during proliferation. (M) Luciferase assays assessed the effect of miR-23a-5p on the activity of lncDum. (N) Cell count was measured via a CCK-8 assay in C2C12 myoblasts transfected with p-lncDum, NC or p-lncDum and miR-23a-5p mimics. (O) RT-qPCR analysis of the expression levels of Cyclin E, Cyclin B, CCND1 and P21. (P) Bright-field was used to microscopy observe the ability of C2C12 myoblasts to differentiate into myotubes at 6 days after transfecting with p-lncDum, NC or p-lncDum and miR-23a-5p mimics. Scale bar, 100 µm. (Q) RT-qPCR analysis of the expression levels of MyoG, MyoD, MRF4, Myf5 and MyHC. (R) Western blotting analysis of MyoG protein levels in C2C12 myoblasts transfected with miR-23a-5p mimics and p-lncDum, NC or p-lncDum during differentiation. Data are presented as the mean ± SEM of three independent repeats. *P<0.05, **P<0.01. miR, microRNA; lnc, long non-coding RNA; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering; NC, negative control; CCK-8, Cell Counting Kit-8; MyHC, myosin heavy chain; OD, optical density; NS, not significant.

A number of lncRNAs that function in skeletal myogenesis have previously been identified as ceRNAs that sponge miRNAs via complementary base pairing (48,49). In the present study, it was observed that lncDum contained a complementary sequence to that of miR-23a-5p, and that lncDum and miR-23a-5p exhibited opposite mRNA expression level patterns during C2C12 myoblast proliferation and differentiation (Fig. 4J and K). Moreover, RT-qPCR analysis demonstrated that overexpression of miR-23a-5p significantly inhibited the increase of lncDum induced by transfection of p-lncDum (Fig. 4L). A luciferase reporter assay demonstrated that, compared with the control, overexpression of miR-23a-5p suppressed the luciferase activity of WT-lncDum, but had little effect on miR-23a-5p binding sites in Mut-lncDum (Fig. 4M), demonstrating that miR-23a-5p interacted with lncDum via complementary base pairing. Moreover, rescue experiments were performed to evaluate whether the effects of miR-23a-5p on the proliferation and differentiation of C2C12 myoblasts were regulated by lncDum. The results indicated that miR-23a-5p significantly attenuated the effect of lncDum overexpression on C2C12 myoblast proliferation and differentiation (Fig. 4N-R). Taken together, these data suggested that miR-23a-5p mediated myogenic proliferation and differentiation via interactions with lncDum.

Discussion

Skeletal myogenesis is a complex biological process (7). In addition to the involvement of myoblast proliferation and differentiation, differentiated myoblasts fuse into multinucleate myotubes, which give rise to diverse types of muscle fiber that build the complex skeletal muscle architecture essential for body movement, postural behavior and breathing (1,50). Previous studies have also demonstrated the roles of miRNAs in skeletal myogenesis. For example, Naguibneva et al (42) reported that miR-181 regulates mammalian myoblast differentiation via targeting homeobox protein Hox-A11. Mi et al (30) reported that miR-139-5p regulates C2C12 cell myogenesis via blocking the Wnt/β-catenin signaling pathway. Wang et al (10) reported that miR-23a-3p regulates myogenic differentiation by inhibiting the expression levels of fast MyHC isoforms. To the best of our knowledge, however, the role and molecular mechanism of miR-23a-5p in myoblast proliferation and differentiation has not previously been fully elucidated. The present study aimed to determine whether miR-23a-5p was involved in skeletal myogenesis. Previous studies have indicated that miR-206-3p (41), miR-181a-5p (42) and miR-221 (43) serve key roles in the regulation of skeletal myogenesis. The results of the present study demonstrated that miR-23a-5p exhibited a similar expression level pattern to these three myoblast-specific miRNAs. The present study also revealed that miR-23a-5p was ubiquitously expressed in different types of tissue and moderately expressed in muscle tissue. Furthermore, miR-23a-5p expression levels in muscle tissue were significantly decreased following Dex treatment. Dex, a potent synthetic glucocorticoid, has been widely used to induce muscle atrophy due to its ability to stimulate protein degradation (51). Muscle atrophy induced by muscular dysfunction can affect functional capacity. The data obtained in the present study supported the hypothesis that miR-23a-5p is associated with skeletal myogenesis. As miR-23a-5p exhibited low expression during the proliferation and differentiation of C2C12 myoblasts compared with myogenesis-specific miRNAs, including miR-206-3p, miR-181a-5p, miR-221/222 and miR-29b-3p, it was hypothesized that overexpression of miR-23a-5p could affect skeletal myogenesis.

Myoblast proliferation and differentiation are key processes in skeletal myogenesis (7). Cyclin E, Cyclin B and CCND1, which bind cyclin-dependent protein kinases (Cdk) to control cell cycle progression, such as G1-S and G2-M transition, are expressed during the course of the cell cycle (52–54). By contrast, as a Cdk inhibitor, p21 can bind and inactivate Cdk-cyclin complexes to repress specific steps of cell cycle progression (55–57). The results of the present study demonstrated that miR-23a-5p positively regulate the expression levels of these genes during C2C12 myoblast proliferation. EdU and CCK-8 assays also showed that miR-23a-5p significantly promoted C2C12 myoblast proliferation.

Previous studies have shown that myogenic differentiation is controlled by complex myogenic transcription factors, such as MyoD, Myf5, MyoG, MRF4 and MyHC (58,59). MyoD and Myf5 participate in controlling early differentiation, and MyoG and MRF4 induce myoblast differentiation at later stages (30,42). MyoG and MyoD are key transcription factors in myogenesis and regulate transcription of the majority of muscle-specific genes (60). MyoG and MyoD serve an important role in the regulation of myoblast differentiation. MyoD is considered to act as a myogenic determination gene (61,62), whereas MyoG is essential for terminal differentiation of committed myoblasts (59). The present results indicated that miR-23a-5p decreased the expression levels of myogenesis-specific factors and that miR-23a-5p inhibits C2C12 myoblast differentiation. Previous studies have demonstrated that regulators could modulate muscle fiber type transition via altering the percentage of MyHC isoforms associated with slow- or fast-twitch fibers (63–65). Fibers expressing MyHC2a and 2× have intermediate characteristics between MyHC types 1 and 2b (66). In addition, muscle fiber type composition is key for muscle biological function and body metabolism; therefore, the present study aimed to determine whether miR-23a-5p mediates muscle fiber type composition. The results suggested that miR-23a-5p is involved in regulating the composition of skeletal muscle fiber type in a more complex manner. Taken together, these results indicated that miR-23a-5p may affect C2C12 myoblast differentiation and mediate skeletal muscle fiber-type composition.

A number of lncRNAs that are key regulators of skeletal muscle physiology and are involved in skeletal myogenesis have been identified as ceRNAs that sponge miRNAs via complementary base pairing (34,67). Wang et al (47) indicated that lncDum serves an important role in regulating cell differentiation and muscle regeneration. These results suggest that miR-23a-5p interacted with lncDum by complementary base pairing, and that lncDum promotes C2C12 myoblast proliferation and differentiation. Taken together, these data indicated that miR-23a-5p mediates myogenic proliferation and differentiation via interacting with lncDum.

In conclusion, the present study demonstrated that the expression levels of miR-23a-5p were showed a dynamic change, from decrease to increase, during myogenesis in mice. Analysis revealed that miR-23a-5p overexpression promoted C2C12 myoblast proliferation, inhibited C2C12 myoblast differentiation and regulated muscle fiber type composition. The present study indicated that lncDum affected C2C12 myoblast proliferation and differentiation, which was contrary to the effects of miR-23a-5p. Taken together, the present results suggested that lncDum may be a target of miR-23a-5p in the regulation of skeletal myogenesis. However, the association between miR-23a-5p inhibition and the regulation of skeletal myogenesis was not determined in the present study, and further investigation is required to elucidate the regulatory mechanisms underlying miR-23a-5p in skeletal muscle development.