miR-152 regulates the proliferation and differentiation of C2C12 myoblasts by targeting <Emphasis Type="Italic">E2F3</Emphasis>

The development of skeletal muscle is a complex process involving the proliferation, differentiation, apoptosis, and changing of muscle fiber types in myoblasts. Many reports have described the involvement of microRNAs in the myogenesis of myoblasts. In this study, we found that the expression of miR-152 was gradually down-regulated during myoblast proliferation, but gradually up-regulated during the differentiation of myoblasts. Transfection with miR-152 mimics restrained cell proliferation and decreased the expression levels of cyclin E, CDK4, and cyclin D1, but promoted myotube formation and significantly increased the mRNA expression levels of MyHC, MyoD, MRF4, and MyoG in C2C12 myoblasts. However, treatment with miR-152 inhibitors promoted cell proliferation and restrained differentiation. Moreover, over-expression of miR-152 significantly decreased E2F3 production in C2C12 myoblasts. A luciferase assay confirmed that miR-152 could bind to the 3′ UTR of E2F3. In conclusion, this study showed that miR-152 inhibited proliferation and promoted myoblast differentiation by targeting E2F3.     

miR-22 regulates C2C12 myoblast proliferation and differentiation by targeting TGFBR1

Recently, miR-22 was found to be differentially expressed in different skeletal muscle growth period, indicated that it might have function in skeletal muscle myogenesis. In this study, we found that the expression of miR-22 was the most in skeletal muscle and was gradually up-regulated during mouse myoblast cell (C2C12 myoblast cell line) differentiation. Overexpression of miR-22 repressed C2C12 myoblast proliferation and promoted myoblast differentiation into myotubes, whereas inhibition of miR-22 showed the opposite results. During myogenesis, we predicted and verified transforming growth factor beta receptor 1 (TGFBR1), a key receptor of the TGF-β/Smad signaling pathway, was a target gene of miR-22. Then, we found miR-22 could regulate the expression of TGFBR1 and down-regulate the Smad3 signaling pathway. Knockdown of TGFBR1 by siRNA suppressed the proliferation of C2C12 cells but induced its differentiation. Conversely, overexpression of TGFBR1 significantly promoted proliferation but inhibited differentiation of the myoblast. Additionally, when C2C12 cells were treated with different concentrations of transforming growth factor beta 1 (TGF-β1), the level of miR-22 in C2C12 cells was reduced. The TGFBR1 protein level was significantly elevated in C2C12 cells treated with TGF-β1. Moreover, miR-22 was able to inhibit TGF-β1-induced TGFBR1 expression in C2C12 cells. Altogether, we demonstrated that TGF-β1 inhibited miR-22 expression in C2C12 cells and miR-22 regulated C2C12 cell myogenesis by targeting TGFBR1.     

miR-143-3p促进C2C12成肌细胞分化

MicroRNAs (miRNAs) 是一类小非编码RNA,近年研究发现其在骨骼肌发育调控中发挥重要作用.为探明miR-143-3p在C2C12成肌细胞分化中的调控作用,采用 real-time PCR 检测了miR-143-3p在小鼠各组织及C2C12成肌细胞分化过程中的表达;使用miR-143-3p 的模拟物和特异性抑制剂分别处理细胞,采用 real-time PCR 和 Western印迹分别检测成肌因子 MyoG和成肌标志基因 MyHC mRNA和蛋白水平的变化;用免疫荧光染色的方法观察肌管的形成.结果显示,miR-143-3p在小鼠各组织中均有表达,并且随着细胞分化表达量逐渐增加;C2C12成肌细胞过表达 miR-143-3p,与对照组相比,成肌调控因子MyoG和成肌标志基因MyHC 的mRNA和蛋白表达均显著升高,肌管数量明显增多;抑制剂处理结果显示,细胞分化被显著抑制.检测miR-143-3p对MyHC各亚型表达的影响发现,miR-143-3p表达的变化并不直接影响MyHC各亚型的表达.以上结果说明, miR-143-3p在骨骼肌和成肌细胞中均有表达,能够促进C2C12成肌细胞分化,但并不直接调控MyHCs的表达.     

MicroRNA-27a promotes myoblast proliferation by targeting myostatin

MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. However, the role of miRNAs in myoblast proliferation remains poorly understood. Here we found that the expression of miR-27a was increased during proliferation of C2C12 myoblasts. Moreover, overexpression of miR-27a in C2C12 cells promoted myoblast proliferation by reducing the expression of myostatin, a critical inhibitor of skeletal myogenesis. In addition, the miR-27a was confirmed to target myostatin 3'UTR by a luciferase reporter analysis. Together, these results suggest that miR-27a promotes myoblast proliferation through targeting myostatin.     

Transient production of alpha-smooth muscle actin by skeletal myoblasts during differentiation in culture and following intramuscular implantation

alpha-smooth muscle actin (SMA) is typically not present in post-embryonic skeletal muscle myoblasts or skeletal muscle fibers. However, both primary myoblasts isolated from neonatal mouse muscle tissue, and C2C12, an established myoblast cell line, produced SMA in culture within hours of exposure to differentiation medium. The SMA appeared during the cells' initial elongation, persisted through differentiation and fusion into myotubes, remained abundant in early myotubes, and was occasionally observed in a striated pattern. SMA continued to be present during the initial appearance of sarcomeric actin, but disappeared shortly thereafter leaving only sarcomeric actin in contractile myotubes derived from primary myoblasts. Within one day after implantation of primary myoblasts into mouse skeletal muscle, SMA was observed in the myoblasts; but by 9 days post-implantation, no SMA was detectable in myoblasts or muscle fibers. Thus, both neonatal primary myoblasts and an established myoblast cell line appear to similarly reprise an embryonic developmental program during differentiation in culture as well as differentiation within adult mouse muscles.     

The microRNA-127-3p directly targeting Vamp2 in C2C12 myoblasts

MicroRNAs (miRNAs) have been reported that can regulate skeletal muscle growth and development. Previously, we demonstrated that miR-127-3p were differently expressed in skeletal muscle and muscle cells. However, the molecular mechanism of miR-127-3p regulation of skeletal myogenesis are not well elucidated. In this study, we transfected miR-127-3p into C2C12 cells, and found miR-127-3p induces myogenesis by targeting Vamp2. Moreover, the regulatory mechanism of Vamp2 in myoblasts proliferation and differentiation was further confirmed. In conclusion, our data providedevidences that miR-127-3p reciprocally regulated myoblasts proliferation and differentiation through directly targeting Vamp2.     

circUBE3C modulates myoblast development by binding to miR-191 and upregulating the expression of p27

Noncoding RNAs, including miRNAs (microRNAs) and circRNAs (circular RNA), are crucial regulators of myoblast proliferation and differentiation during muscle development. However, the specific roles and molecular mechanisms of circRNAs in muscle development remain poorly understood. Based on the existing circRNA-miRNA-mRNA network, our study focuses on circUBE3C, exploring its differential expression in fetal and adult muscle tissue of the cattle and investigating its impact on myoblast proliferation, apoptosis, and differentiation. The functional analysis of overexpression plasmids and siRNAs (small interfering RNAs) targeting circUBE3C was comprehensively evaluated by employing an array of advanced assays, encompassing CCK-8 (cell counting kit-8), EdU (5-ethynyl-20-deoxyuridine), flow cytometry, western blot analysis, and RT-qPCR. In vivo investigations indicated that overexpression of circUBE3C impedes the process of skeletal muscle regeneration. Mechanistically, we demonstrated that circUBE3C interacts with miR-191 and alleviates the suppression of p27 through cytoplasmic separation, bioinformatics prediction, dual-luciferase reporter assay, and RIP (RNA immunoprecipitation). Our findings indicate that the novel circRNA circUBE3C competitively binds to miR-191, thereby inhibiting proliferation and promoting apoptosis in bovine primary myoblasts and unveiling a regulatory pathway in bovine skeletal muscle development. These findings expand our understanding of circRNA functions in mammals and provide a basis for further exploration of their role in myogenesis and muscle diseases.     

MicroRNA-323-3p promotes myogenesis by targeting Smad2

Skeletal muscle is an important and complex organ with multiple biological functions in humans and animals. Proliferation and differentiation of myoblasts are the key steps during the development of skeletal muscle. MicroRNA (miRNA) is a class of 21-nucleotide noncoding RNAs regulating gene expression by combining with the 3′-untranslated region of target messenger RNA. Many studies in recent years have suggested that miRNAs play a critical role in myogenesis. Through high-throughput sequencing, we found that miR-323-3p showed significant changes in the longissimus dorsi muscle of Rongchang pigs in different age groups. In this study, we discovered that overexpression of miR-323-3p repressed myoblast proliferation and promoted differentiation, whereas the inhibitor of miR-323-3p displayed the opposite results. Furthermore, we predicted Smad2 as the target gene of miR-323-3p and found that miR-323-3p directly modulated the expression level of Smad2. Then luciferase reporter assays verified that Smad2 was a target gene of miR-323-3p during the differentiation of myoblasts. These findings reveal that miR-323-3p is a positive regulator of myogenesis by targeting Smad2. This provides a novel mechanism of miRNAs in myogenesis.     

Recombinant platelet-derived growth factor-BB stimulates growth and inhibits differentiation of rat L6 myoblasts.

We previously found that L6 myoblasts and skeletal muscle isolated from developing rats express the platelet-derived growth factor (PDGF) beta-receptor gene (Jin, P., Rahm, M., Claesson-Welsh, L., Heldin, C.-H., and Sejersen, T. (1990) J. Cell Biol. 110, 1665-1672). We now report that recombinant human PDGF-BB is a mitogen for L6 myoblasts and also a potent inhibitor of myogenic differentiation. Treatment of L6J1 myoblasts with PDGF-BB increased the rate of DNA synthesis and stimulated cell proliferation. In differentiation medium (Dulbecco's modified Eagle's medium/0.5% fetal calf serum or Dulbecco's modified Eagle's medium/insulin), PDGF-BB prevented fusion of confluent myoblasts and suppressed biochemical differentiation in L6J1 cells. Inhibition of myoblast differentiation was, however, reversible. Withdrawal of PDGF-BB from the medium allowed myoblast fusion to occur. Northern blot hybridization showed that the PDGF beta-receptor mRNA was down-regulated to an undetectable level when confluent cultures of L6J1 myoblasts in growth medium (Dulbecco's modified Eagle's medium/5% fetal calf serum) were shifted to differentiation medium. Receptor binding assays further indicated that binding of PDGF-BB to its receptors on L6J1 myoblasts declined rapidly before creatine kinase activity rose. Our results provide the first demonstration that PDGF-BB is a potent regulator of myogenesis of L6 rat myoblasts and suggest that it may regulate muscle differentiation in vivo.     

干扰Sema7A抑制C2C12成肌细胞的增殖和分化

为研究脑信号蛋白家族（Semaphorins）成员Sema7A对成肌细胞增殖和分化的影响,本文设计并合成了Sema7A基因的小干扰RNA（small interfering RNA,siRNA）,用此siRNA转染C2C12成肌细胞．通过Hoechst核染和流式细胞术检测细胞增殖情况,免疫荧光检测肌管的形成情况,real-time qPCR和Western印迹技术检测成肌标记基因的变化.结果显示,干扰Sema7A后,C2C12成肌细胞增殖减慢,处在G2和S期的细胞所占的比例明显下降,而G1期细胞的比例升高．免疫荧光检测结果显示,干扰Sema7A后,肌管的直径及MyHC+细胞所占比例均显著降低．Real-time qPCR和Western印迹结果也显示,肌肉分化标志基因MyoD、MyoG、MyHC的mRNA及蛋白质表达均下降．进一步检测Sema7A受体下游信号通路发现,干扰Sema7A后,其下游信号分子PI3K和AKT的磷酸化水平被下调．以上结果表明,Sema7A可以调节C2C12成肌细胞的增殖和分化,可能是通过其受体作用于PI3K/AKT信号通路实现的,这为进一步研究Sema7A在骨骼肌发育中的作用提供实验基础.     

Stac3 Inhibits Myoblast Differentiation into Myotubes

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.     

KDM4A regulates myogenesis by demethylating H3K9me3 of myogenic regulatory factors

Histone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.Subject terms: Differentiation, Muscle stem cells, Epigenetics     

Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System

Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.     

Isoproterenol induces an increase in muscle fiber size by the proliferation of Pax7‐positive cells and in a mTOR‐independent mechanism

β‐Adrenergic signaling regulates many physiological processes in skeletal muscles. A wealth of evidence has shown that β‐agonists can increase skeletal muscle mass in vertebrates. Nevertheless, to date, the specific role of β‐adrenergic receptors in different cell phenotypes (myoblasts, fibroblasts, and myotubes) and during the different steps of embryonic skeletal muscle differentiation has not been studied. Therefore, here we address this question through the analysis of embryonic chick primary cultures of skeletal muscle cells during the formation of multinucleated myotubes. We used isoproterenol (ISO), a β‐adrenergic receptor agonist, to activate the β‐adrenergic signaling and quantified several aspects of muscle differentiation. ISO induced an increase in myoblast proliferation, in the percentage of Pax7‐positive myoblasts and in the size of skeletal muscle fibers, suggesting that ISO activates a hyperplasic and hypertrophic muscle response. Interestingly, treatment with ISO did not alter the number of fibroblast cells, suggesting that ISO effects are specific to muscle cells in the case of chick myogenic cell culture. We also show that rapamycin, an inhibitor of the mammalian target of rapamycin signaling pathway, did not prevent the effects of ISO on chick muscle fiber size. The collection of these results provides new insights into the role of β‐adrenergic signaling during skeletal muscle proliferation and differentiation and specifically in the regulation of skeletal muscle hyperplasia and hypertrophy.