MicroRNA-27b-3p Targets the Myostatin Gene to Regulate Myoblast Proliferation and Is Involved in Myoblast Differentiation

microRNAs play an important role in the growth and development of chicken embryos, including the regulation of skeletal muscle genesis, myoblast proliferation, differentiation, and apoptosis. Our previous RNA-seq studies showed that microRNA-27b-3p (miR-27b-3p) might play an important role in regulating the proliferation and differentiation of chicken primary myoblasts (CPMs). However, the mechanism of miR-27b-3p regulating the proliferation and differentiation of CPMs is still unclear. In this study, the results showed that miR-27b-3p significantly promoted the proliferation of CPMs and inhibited the differentiation of CPMs. Then, myostatin (MSTN) was confirmed to be the target gene of miR-27b-3p by double luciferase reporter assay, RT-qPCR, and Western blot. By overexpressing and interfering with MSTN expression in CPMs, the results showed that overexpression of MSTN significantly inhibited the proliferation and differentiation of CPMs. In contrast, interference of MSTN expression had the opposite effect. This study showed that miR-27b-3p could promote the proliferation of CPMs by targeting MSTN. Interestingly, both miR-27b-3p and MSTN can inhibit the differentiation of CPMs. These results provide a theoretical basis for further understanding the function of miR-27b-3p in chicken and revealing its regulation mechanism on chicken muscle growth.

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JAK1–STAT1–STAT3, a key pathway promoting proliferation and preventing premature differentiation of myoblasts

The Journal of Cell Biology ◽

10.1083/jcb.200703184 ◽

2007 ◽

Vol 179 (1) ◽

pp. 129-138 ◽

Cited By ~ 113

Author(s):

Luguo Sun ◽

Kewei Ma ◽

Haixia Wang ◽

Fang Xiao ◽

Yan Gao ◽

...

Keyword(s):

Myogenic Differentiation ◽

Muscle Growth ◽

Janus Kinase ◽

Myoblast Differentiation ◽

Muscle Stem Cell ◽

Janus Kinase 1 ◽

Binding Factor ◽

Proliferation And Differentiation ◽

Myoblast Proliferation ◽

Concomitant Reduction

Skeletal muscle stem cell–derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced muscle regeneration. However, the cellular signaling pathways controlling the proliferation and differentiation of myoblasts are not fully understood. We demonstrate that Janus kinase 1 (JAK1) is required for myoblast proliferation and that it also functions as a checkpoint to prevent myoblasts from premature differentiation. Deliberate knockdown of JAK1 in both primary and immortalized myoblasts induces precocious myogenic differentiation with a concomitant reduction in cell proliferation. This is caused, in part, by an accelerated induction of MyoD, myocyte enhancer–binding factor 2 (MEF2), p21Cip1, and p27Kip1, a faster down-regulation of Id1, and an increase in MEF2-dependent gene transcription. Downstream of JAK1, of all the signal transducer and activator of transcriptions (STATs) present in myoblasts, we find that only STAT1 knockdown promotes myogenic differentiation in both primary and immortalized myoblasts. Leukemia inhibitory factor stimulates myoblast proliferation and represses differentiation via JAK1–STAT1–STAT3. Thus, JAK1–STAT1–STAT3 constitutes a signaling pathway that promotes myoblast proliferation and prevents premature myoblast differentiation.

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Male-Biased gga-miR-2954 Regulates Myoblast Proliferation and Differentiation of Chicken Embryos by Targeting YY1

Genes ◽

10.3390/genes12091325 ◽

2021 ◽

Vol 12 (9) ◽

pp. 1325

Author(s):

Xiuxue Dong ◽

Yu Cheng ◽

Lingyun Qiao ◽

Xin Wang ◽

Cuiping Zeng ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Development ◽

Expression Profiles ◽

Myoblast Differentiation ◽

Mammalian Skeletal Muscle ◽

Loss Of Function ◽

Skeletal Muscle Development ◽

Chicken Muscle ◽

Proliferation And Differentiation ◽

Myoblast Proliferation

Previous studies have shown that gga-miR-2954 was highly expressed in the gonads and other tissues of male chickens, including muscle tissue. Yin Yang1 (YY1), which has functions in mammalian skeletal muscle development, was predicted to be a target gene of gga-miR-2954. The purpose of this study was to investigate whether gga-miR-2954 plays a role in skeletal muscle development by targeting YY1, and evaluate its function in the sexual dimorphism development of chicken muscle. Here, all the temporal and spatial expression profiles in chicken embryonic muscles showed that gga-miR-2954 is highly expressed in males and mainly localized in cytoplasm. Gga-miR-2954 exhibited upregulated expression of in vitro myoblast differentiation stages. Next, through the overexpression and loss-of-function experiments performed in chicken primary myoblasts, we found that gga-miR-2954 inhibited myoblast proliferation but promoted differentiation. During myogenesis, gga-miR-2954 could suppress the expression of YY1, which promoted myoblast proliferation and inhibited the process of myoblast cell differentiation into multinucleated myotubes. Overall, these findings reveal a novel role of gga-miR-2954 in skeletal muscle development through its function of the myoblast proliferation and differentiation by suppressing the expression of YY1. Moreover, gga-miR-2954 may contribute to the sex difference in chicken muscle development.

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Anemic Hypoxemia Reduces Myoblast Proliferation and Muscle Growth in Late Gestation Fetal Sheep

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00342.2020 ◽

2021 ◽

Author(s):

Paul J. Rozance ◽

Stephanie R Wesolowski ◽

Sonnet S. Jonker ◽

Laura D Brown

Keyword(s):

Skeletal Muscle ◽

Mrna Expression ◽

Muscle Growth ◽

Late Gestation ◽

Whole Body ◽

Myoblast Differentiation ◽

Fetal Sheep ◽

Body Protein ◽

Skeletal Muscle Growth ◽

Myoblast Proliferation

Fetal skeletal muscle growth requires myoblast proliferation, differentiation, and fusion into myofibers in addition to protein accretion for fiber hypertrophy. Oxygen is an important regulator of this process. Therefore, we hypothesized that fetal anemic hypoxemia would inhibit skeletal muscle growth. Studies were performed in late gestation fetal sheep that were bled to anemic, and therefore hypoxemic, conditions beginning at ~125 days of gestation (term = 148 days) for 9 ± 0 days (n=19) and compared to control fetuses (n=16). A metabolic study was performed on gestational day ~134 to measure fetal protein kinetic rates. Myoblast proliferation and myofiber area were determined in biceps femoris (BF), tibialis anterior (TA), and flexor digitorum superficialis (FDS) muscles. mRNA expression of muscle regulatory factors was determined in BF. Fetal arterial hematocrit and oxygen content were 28% and 52% lower, respectively, in anemic fetuses. Fetal weight and whole-body protein synthesis, breakdown, and accretion rates were not different between groups. Hindlimb length, however, was 7% shorter in anemic fetuses. TA and FDS muscles weighed less and FDS myofiber area was smaller in anemic fetuses compared to controls. The percentage of Pax7+ myoblasts that expressed Ki67 was lower in BF and tended to be lower in FDS from anemic fetuses indicating reduced myoblast proliferation. There was less MYOD and MYF6 mRNA expression in anemic vs. control BF consistent with reduced myoblast differentiation. These results indicate that fetal anemic hypoxemia reduced muscle growth. We speculate that fetal muscle growth may be improved by strategies that increase oxygen availability.

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MiR-34b-5p Mediates the Proliferation and Differentiation of Myoblasts by Targeting IGFBP2

Cells ◽

10.3390/cells8040360 ◽

2019 ◽

Vol 8 (4) ◽

pp. 360 ◽

Cited By ~ 4

Author(s):

Wang ◽

Zhang ◽

Li ◽

Abdalla ◽

Chen ◽

...

Keyword(s):

Skeletal Muscle ◽

Cell Cycle Progression ◽

Muscle Development ◽

Muscle Growth ◽

Flow Cytometric Analysis ◽

Luciferase Reporter ◽

Cycle Progression ◽

Flow Cytometric ◽

Proliferation And Differentiation ◽

Dual Luciferase Reporter Assay

As key post-transcriptional regulators, microRNAs (miRNAs) play an indispensable role in skeletal muscle development. Our previous study suggested that miR-34b-5p and IGFBP2 could have a potential role in skeletal muscle growth. Our goal in this study is to explore the function and regulatory mechanism of miR-34b-5p and IGFBP2 in myogenesis. In this study, the dual-luciferase reporter assay and Western blot analysis showed that IGFBP2 is a direct target of miR-34b-5p. Flow cytometric analysis and EdU assay showed that miR-34b-5p could repress the cell cycle progression of myoblasts, and miR-34b-5p could promote the formation of myotubes by promoting the expression of MyHC. On the contrary, the overexpression of IGFBP2 significantly facilitated the proliferation of myoblasts and hampered the formation of myotubes. Together, our results indicate that miR-34b-5p could mediate the proliferation and differentiation of myoblasts by targeting IGFBP2.

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miR-194-5p negatively regulates the proliferation and differentiation of rabbit skeletal muscle satellite cells

Molecular and Cellular Biochemistry ◽

10.1007/s11010-020-03918-0 ◽

2020 ◽

Author(s):

Yu Shi ◽

Xudong Mao ◽

Mingcheng Cai ◽

Shenqiang Hu ◽

Xiulan Lai ◽

...

Keyword(s):

Skeletal Muscle ◽

Satellite Cells ◽

Mammalian Species ◽

Muscle Growth ◽

Luciferase Reporter ◽

Stem Cell Population ◽

Muscle Satellite Cells ◽

Skeletal Muscle Satellite Cells ◽

Proliferation And Differentiation

Abstract Skeletal muscle satellite cells (SMSCs), also known as a multipotential stem cell population, play a crucial role during muscle growth and regeneration. In recent years, numerous miRNAs have been associated with the proliferation and differentiation of SMSCs in a number of mammalian species; however, the regulatory mechanisms of miR-194-5p in rabbit SMSCs still remain scarce. In this study, miR-194-5p was first observed to be highly expressed in the rabbit leg muscle. Furthermore, both the mimics and inhibitor of miR-194-5p were used to explore its role in the proliferation and differentiation of rabbit SMSCs cultured in vitro. Results from both EdU and CCK8 assays showed that miR-194-5p inhibited the proliferation of SMSCs. Meanwhile, Mef2c was identified as a target gene of miR-194-5p based on the dual-luciferase reporter assay results. In addition, upregulation of miR-194-5p decreased the expression levels of Mef2c and MyoG during rabbit SMSCs differentiation on Days 3 and 7 of in vitro culture. Taken together, these data demonstrated that miR-194-5p negatively regulates the proliferation and differentiation of rabbit SMSCs by targeting Mef2c.

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Thyroid Hormone Receptor α Plays an Essential Role in Male Skeletal Muscle Myoblast Proliferation, Differentiation, and Response to Injury

Endocrinology ◽

10.1210/en.2015-1443 ◽

2016 ◽

Vol 157 (1) ◽

pp. 4-15 ◽

Cited By ~ 21

Author(s):

Anna Milanesi ◽

Jang-Won Lee ◽

Nam-Ho Kim ◽

Yan-Yun Liu ◽

An Yang ◽

...

Keyword(s):

Skeletal Muscle ◽

Thyroid Hormone ◽

Hormone Receptor ◽

Essential Role ◽

Mutant Mouse ◽

Thyroid Hormone Receptor ◽

Myogenic Differentiation ◽

Myoblast Differentiation ◽

Primary Myoblasts ◽

Myoblast Proliferation

Abstract Thyroid hormone plays an essential role in myogenesis, the process required for skeletal muscle development and repair, although the mechanisms have not been established. Skeletal muscle develops from the fusion of precursor myoblasts into myofibers. We have used the C2C12 skeletal muscle myoblast cell line, primary myoblasts, and mouse models of resistance to thyroid hormone (RTH) α and β, to determine the role of thyroid hormone in the regulation of myoblast differentiation. T3, which activates thyroid hormone receptor (TR) α and β, increased myoblast differentiation whereas GC1, a selective TRβ agonist, was minimally effective. Genetic approaches confirmed that TRα plays an important role in normal myoblast proliferation and differentiation and acts through the Wnt/β-catenin signaling pathway. Myoblasts with TRα knockdown, or derived from RTH-TRα PV (a frame-shift mutation) mice, displayed reduced proliferation and myogenic differentiation. Moreover, skeletal muscle from the TRα1PV mutant mouse had impaired in vivo regeneration after injury. RTH-TRβ PV mutant mouse model skeletal muscle and derived primary myoblasts did not have altered proliferation, myogenic differentiation, or response to injury when compared with control. In conclusion, TRα plays an essential role in myoblast homeostasis and provides a potential therapeutic target to enhance skeletal muscle regeneration.

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Regulation of myostatin expression and myoblast differentiation by FoxO and SMAD transcription factors

AJP Cell Physiology ◽

10.1152/ajpcell.00542.2005 ◽

2007 ◽

Vol 292 (1) ◽

pp. C188-C199 ◽

Cited By ~ 154

Author(s):

David L. Allen ◽

Terry G. Unterman

Keyword(s):

Transcription Factors ◽

Promoter Activity ◽

Transforming Growth Factor ◽

Active Form ◽

Muscle Growth ◽

Myoblast Differentiation ◽

Binding Assays ◽

Myostatin Gene

Myostatin, a member of the transforming growth factor (TGF)-β family, plays an important role in regulating skeletal muscle growth and differentiation. Here we examined the role of FoxO1 and SMAD transcription factors in regulating myostatin gene expression and myoblast differentiation in C2C12 myotubes in vitro. Both myostatin and FoxO1 mRNA expression were greater in fast- vs. slow-twitch skeletal muscles in vivo. Moreover, expression of a constitutively active form of FoxO1 increased myostatin mRNA and increased activity of a myostatin promoter reporter construct in differentiated C2C12 myotubes. Mutagenesis of highly conserved FoxO or SMAD binding sites significantly decreased myostatin promoter activity, and binding assays showed that both FoxO1 and SMADs bind to their respective sites in the myostatin promoter. Treatment with TGF-β and/or overexpression of SMAD2, -3, or -4 also resulted in a significant increase in myostatin promoter activity. Treatment with TGF-β along with overexpression of SMAD2 and FoxO1 resulted in the largest increase in myostatin promoter activity. Finally, TGF-β treatment and SMAD2 overexpression greatly potentiated FoxO1-mediated suppression of myoblast differentiation. Together these data demonstrate that FoxO1 and SMAD transcription factors regulate the expression of myostatin and contribute to the control of muscle cell growth and differentiation.

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The Effects of Marine Algal Polyphenols, Phlorotannins, on Skeletal Muscle Growth in C2C12 Muscle Cells via Smad and IGF-1 Signaling Pathways

Marine Drugs ◽

10.3390/md19050266 ◽

2021 ◽

Vol 19 (5) ◽

pp. 266

Author(s):

Seo-Young Kim ◽

Ji-Hyeok Lee ◽

Nalae Kang ◽

Kil-Nam Kim ◽

You-Jin Jeon

Keyword(s):

Skeletal Muscle ◽

Muscle Growth ◽

Negative Regulator ◽

Growth Effect ◽

C2c12 Myoblast ◽

Synthetic Drugs ◽

Proliferation And Differentiation ◽

Myoblast Proliferation ◽

C2c12 Muscle Cells ◽

Marine Algal

Skeletal muscle is an important tissue in energy metabolism and athletic performance. The use of effective synthetic supplements and drugs to promote muscle growth is limited by various side effects. Moreover, their use is prohibited by anti-doping agencies; hence, natural alternatives are needed. Therefore, we evaluated the muscle growth effect of substances that can act like synthetic supplements from edible marine algae. First, we isolated six marine algal polyphenols belonging to the phlorotannin class, namely dieckol (DK), 2,7′′-phloroglucinol-6,6′-bieckol (PHB), phlorofucofuroeckol A (PFFA), 6,6′-bieckol (6,6-BK), pyrogallol-phloroglucinol-6,6′-bieckol (PPB), and phloroglucinol (PG) from an edible brown alga, Ecklonia cava and evaluated their effects on C2C12 myoblasts proliferation and differentiation. Of the six phlorotannin isolates evaluated, DK and PHB induced the highest degree of C2C12 myoblast proliferation. In addition, DK and PHB regulates myogenesis by down-regulating the Smad signaling, a negative regulator, and up-regulating the insulin-like growth factor-1 (IGF-1) signaling, a positive regulator. Interestingly, DK and PHB bind strongly to myostatin, which is an inhibitor of myoblast proliferation, while also binding to IGF-1 receptors. Moreover, they bind to IGF-1 receptor. These results suggest that DK and PHB are potential natural muscle building supplements and could be a safer alternative to synthetic drugs.

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Impact of vitamin D and vitamin D receptor TaqI polymorphism in primary human myoblasts

Endocrine Connections ◽

10.1530/ec-19-0194 ◽

2019 ◽

Vol 8 (7) ◽

pp. 1070-1081

Author(s):

Amarjit Saini ◽

Linda Björkhem-Bergman ◽

Johan Boström ◽

Mats Lilja ◽

Michael Melin ◽

...

Keyword(s):

Vitamin D ◽

Vitamin D Receptor ◽

Cellular Response ◽

Myoblast Differentiation ◽

Differentiation Markers ◽

Vdr Polymorphism ◽

Proliferation And Differentiation ◽

Myoblast Proliferation

The CC genotype of the vitamin D receptor (VDR) polymorphism TaqI rs731236 has previously been associated with a higher risk of developing myopathy compared to TT carriers. However, the mechanistic role of this polymorphism in skeletal muscle is not well defined. The effects of vitamin D on patients genotyped for the VDR polymorphism TaqI rs731236, comparing CC and TT carriers were evaluated. Primary human myoblasts isolated from 4 CC carriers were compared with myoblasts isolated from four TT carriers and treated with vitamin D in vitro. A dose-dependent inhibitory effect on myoblast proliferation and differentiation was observed concurrent with modifications of key myogenic regulatory factors. RNA sequencing revealed a vitamin D dose–response gene signature enriched with a higher number of VDR-responsive elements (VDREs) per gene. Interestingly, the greater the expression of muscle differentiation markers in myoblasts, the more pronounced was the vitamin D-mediated response to suppress genes associated with myogenic fusion and myotube formation. This novel finding provides a mechanistic explanation to the inconsistency regarding previous reports of the role of vitamin D in myoblast differentiation. No effects in myoblast proliferation, differentiation or gene expression were related to CC vs TT carriers. Our findings suggest that the VDR polymorphism TaqI rs731236 comparing CC vs TT carriers did not influence the effects of vitamin D on primary human myoblasts and that vitamin D inhibits myoblast proliferation and differentiation through key regulators of cell cycle progression. Future studies need to employ strategies to identify the primary responses of vitamin D that drive the cellular response towards quiescence.

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Lnc-GD2H Promotes Proliferation by Forming a Feedback Loop With c-Myc and Enhances Differentiation Through Interacting With NACA to Upregulate Myog in C2C12 Myoblasts

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.671857 ◽

2021 ◽

Vol 9 ◽

Author(s):

Rui Chen ◽

Si Lei ◽

Yanling She ◽

Shanyao Zhou ◽

Huacai Shi ◽

...

Keyword(s):

Muscle Regeneration ◽

Feedback Loop ◽

Quantitative Polymerase Chain Reaction ◽

Cell Counting ◽

Luciferase Reporter ◽

Myoblast Differentiation ◽

Marker Genes ◽

C2c12 Myoblast ◽

Myoblast Proliferation

In the present study, the roles of a novel long non-coding RNA (lncRNA), lnc-GD2H, in promoting C2C12 myoblast proliferation and differentiation and muscle regeneration were investigated by quantitative polymerase chain reaction, western blotting, Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine (EdU), immunofluorescence staining, luciferase reporter, mass spectrometry, pulldown, chromatin immunoprecipitation, RNA immunoprecipitation assay, wound healing assays, and cardiotoxin (CTX)-induced muscle injury assays. It was observed that lnc-GD2H promoted myoblast proliferation as evidenced by the enhancement of the proliferation markers c-Myc, CDK2, CDK4, and CDK6, percentage of EdU-positive cells, and rate of cell survival during C2C12 myoblast proliferation. Additional experiments confirmed that c-Myc bound to the lnc-GD2H promoter and regulated its transcription. lnc-GD2H promoted cell differentiation with enhanced MyHC immunostaining as well as increased expression of the myogenic marker genes myogenin (Myog), Mef2a, and Mef2c during myoblast differentiation. Additional assays indicated that lnc-GD2H interacted with NACA which plays a role of transcriptional regulation in myoblast differentiation, and the enrichment of NACA at the Myog promoter was impaired by lnc-GD2H. Furthermore, inhibition of lnc-GD2H impaired muscle regeneration after CTX-induced injury in mice. lnc-GD2H facilitated the expression of proliferating marker genes and formed a feedback loop with c-Myc during myoblast proliferation. In differentiating myoblasts, lnc-GD2H interacted with NACA to relieve the inhibitory effect of NACA on Myog, facilitating Myog expression to promote differentiation. The results provide evidence for the role of lncRNAs in muscle regeneration and are useful for developing novel therapeutic targets for muscle disorders.

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