Regulates Myoblast Proliferation Research Articles

Pleiotropic Gene HMGA2 Regulates Myoblast Proliferation and Affects Body Size of Sheep.

Uncovering genes associated with muscle growth and body size will benefit the molecular breeding of meat Hu sheep. HMGA2 has proven to be an important gene in mouse muscle growth and is associated with the body size of various species. However, its roles in sheep are still limited. Using sheep myoblast as a cell model, the overexpression of HMGA2 significantly promoted sheep myoblast proliferation, while interference with HMGA2 expression inhibited proliferation, indicated by qPCR, EdU, and CCK-8 assays. Furthermore, the dual-luciferase reporter system indicated that the region NC_056056.1: 154134300-154134882 (-618 to -1200 bp upstream of the HMGA2 transcription start site) was one of the habitats of the HMGA2 core promoter, given the observation that this fragment led to a ~3-fold increase in luciferase activity. Interestingly, SNP rs428001129 (NC_056056.1:g.154134315 C>A) was detected in this fragment by Sanger sequencing of the PCR product of pooled DNA from 458 crossbred sheep. This SNP was found to affect the promoter activity and was significantly associated with chest width at birth and two months old, as well as chest depth at two and six months old. The data obtained in this study demonstrated the phenotypic regulatory role of the HMGA2 gene in sheep production traits and the potential of rs428001129 in marker-assisted selection for sheep breeding in terms of chest width and chest depth.

CMTM3 Suppresses Proliferation and Osteogenic Transdifferentiation of C2C12 Myoblasts through p53 Upregulation.

CKLF-like MARVEL transmembrane domain-containing 3 (CMTM3), a member of the CMTM family that is closely related to tumor occurrence and progression, plays crucial roles in the immune system, cardiovascular system, and male reproductive system. Recently, CMTM3 has emerged as a potential target for treating diseases related to bone formation. However, additional studies are needed to understand the mechanisms by which CMTM3 regulates the process of osteogenic differentiation. In this study, we observed a significant downregulation of Cmtm3 expression during the transdifferentiation of C2C12 myoblasts into osteoblasts induced by BMP4. Cmtm3 overexpression suppressed proliferation and osteogenic differentiation in BMP4-induced C2C12 cells, whereas its knockdown conversely facilitated the process. Mechanistically, Cmtm3 overexpression upregulated both the protein and mRNA levels of p53 and p21. Conversely, Cmtm3 knockdown exerted the opposite effects. Additionally, we found that Cmtm3 interacts with p53 and increases protein stability by inhibiting proteasome-mediated ubiquitination and degradation. Notably, Trp53 downregulation abrogated the inhibitory effect of Cmtm3 on BMP4-induced proliferation and osteogenic differentiation of C2C12 myoblasts. Collectively, our findings provide key insights into the role of CMTM3 in regulating myoblast proliferation and transdifferentiation into osteoblasts, highlighting its significance in osteogenesis research.

Expression of PDLIM5 Spliceosomes and Regulatory Functions on Myogenesis in Pigs

Expression of PDLIM5 Spliceosomes and Regulatory Functions on Myogenesis in Pigs

Anti-apoptotic protein Bcl-2 contributes to the determination of reserve cells during myogenic differentiation of C2C12 cells.

Skeletal muscle's regenerative ability is vital for maintaining muscle function, but chronic diseases like Duchenne muscular dystrophy can deplete this capacity. Muscle satellite cells, quiescent in normal situations, are activated during muscle injury, expressing myogenic regulatory factors, and producing myogenic progenitor cells. It was reported that muscle stem cells in primary culture and reserve cells in C2C12 cells express anti-apoptotic protein Bcl-2. Although the role of Bcl-2 expressed in myogenic cells has been thought to be to enhance cell viability, we hypothesized that Bcl-2 may promote the formation of reserve cells. The expression pattern analysis showed the expression of Bcl-2 in undifferentiated mononucleated cells, emphasizing its usefulness as a reserve cell marker and reminding us that cells expressing Bcl-2 have low proliferative potential. Silencing of Bcl-2 by transfection with siRNA decreased cell viability and the number of reserve cells, while overexpression of Bcl-2 not only increases cell viability but also inhibits muscle differentiation and proliferation. These results emphasize dual roles of Bcl-2 in protecting cells from apoptosis and contributing to reserve cell formation by regulating myoblast proliferation and/or differentiation. Overall, the study sheds light on the multifaceted role of Bcl-2 in the maintenance of skeletal muscle regeneration.

RASGRP1 targeted by H3K27me3 regulates myoblast proliferation and differentiation in mice and pigs.

Skeletal muscle is not only the largest organ in the body that is responsible for locomotion and exercise but also crucial for maintaining the body's energy metabolism and endocrine secretion. The trimethylation of histone H3 lysine 27 (H3K27me3) is one of the most important histone modifications that participates in muscle development regulation by repressing the transcription of genes. Previous studies indicate that the RASGRP1 gene is regulated by H3K27me3 in embryonic muscle development in pigs, but its function and regulatory role in myogenesis are still unclear. In this study, we verify the crucial role of H3K27me3 in RASGRP1 regulation. The gain/loss function of RASGRP1 in myogenesis regulation is performed using mouse myoblast C2C12 cells and primarily isolated porcine skeletal muscle satellite cells (PSCs). The results of qPCR, western blot analysis, EdU staining, CCK-8 assay and immunofluorescence staining show that overexpression of RASGRP1 promotes cell proliferation and differentiation in both skeletal muscle cell models, while knockdown of RASGRP1 leads to the opposite results. These findings indicate that RASGRP1 plays an important regulatory role in myogenesis in both mice and pigs.

LncRNA GTL2 regulates myoblast proliferation and differentiation via the PKA-CREB pathway in Duolang sheep.

Long non-coding RNAs (lncRNAs), which are RNA molecules longer than 200 nucleotides that do not encode proteins, are implicated in a variety of biological processes, including growth and development. Despite research into the role of lncRNAs in skeletal muscle development, the regulatory mechanisms governing ovine skeletal muscle development remain unclear. In this study, we analyzed the expression profiles of lncRNAs in skeletal muscle from 90-day-old embryos (F90), 1-month-old lambs (L30), and 3-year-old adult sheep (A3Y) using RNA sequencing. In total, 4 738 lncRNAs were identified, including 997 that were differentially expressed. Short-time series expression miner analysis identified eight significant expression profiles and a subset of lncRNAs potentially involved in muscle development. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the predicted target genes of these lncRNAs were primarily enriched in pathways associated with muscle development, such as the cAMP and Wnt signaling pathways. Notably, the expression of lncRNA GTL2 was found to decrease during muscle development. Moreover, GTL2 was highly expressed during the differentiation of skeletal muscle satellite cells (SCs) and was shown to modulate ovine myogenesis by affecting the phosphorylation levels of PKA and CREB. Additionally, GTL2 was found to regulate both the proliferation and differentiation of SCs via the PKA-CREB signaling pathway. Overall, this study provides a valuable resource and offers novel insights into the functional roles and regulatory mechanisms of lncRNAs in ovine skeletal muscle growth and development.

Whole-Transcriptome RNA Sequencing Uncovers the Global Expression Changes and RNA Regulatory Networks in Duck Embryonic Myogenesis.

Duck meat is pivotal in providing high-quality protein for human nutrition, underscoring the importance of studying duck myogenesis. The regulatory mechanisms governing duck myogenesis involve both coding and non-coding RNAs, yet their specific expression patterns and molecular mechanisms remain elusive. To address this knowledge gap, we performed expression profiling analyses of mRNAs, lncRNAs, circRNAs, and miRNAs involved in duck myogenesis using whole-transcriptome RNA-seq. Our analysis identified 1733 differentially expressed (DE)-mRNAs, 1116 DE-lncRNAs, 54 DE-circRNAs, and 174 DE-miRNAs when comparing myoblasts and myotubes. A GO analysis highlighted the enrichment of DE molecules in the extracellular region, protein binding, and exocyst. A KEGG analysis pinpointed pathways related to ferroptosis, PPAR signaling, nitrogen metabolism, cell cycle, cardiac muscle contraction, glycerolipid metabolism, and actin cytoskeleton. A total of 51 trans-acting lncRNAs, including ENSAPLT00020002101 and ENSAPLT00020012069, were predicted to participate in regulating myoblast proliferation and differentiation. Based on the ceRNAs, we constructed lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA ceRNA networks involving five miRNAs (miR-129-5p, miR-133a-5p, miR-22-3p, miR-27b-3p, and let-7b-5p) that are relevant to myogenesis. Furthermore, the GO and KEGG analyses of the DE-mRNAs within the ceRNA network underscored the significant enrichment of the glycerolipid metabolism pathway. We identified five different DE-mRNAs, specifically ENSAPLG00020001677, ENSAPLG00020002183, ENSAPLG00020005019, ENSAPLG00020010497, and ENSAPLG00020017682, as potential target genes that are crucial for myogenesis in the context of glycerolipid metabolism. These five mRNAs are integral to ceRNA networks, with miR-107_R-2 and miR-1260 emerging as key regulators. In summary, this study provides a valuable resource elucidating the intricate interplay of mRNA-lncRNA-circRNA-miRNA in duck myogenesis, shedding light on the molecular mechanisms that govern this critical biological process.

Effect of Bovine MEF2A Gene Expression on Proliferation and Apoptosis of Myoblast Cells.

Myocyte enhancer factor 2A (MEF2A) is a member of the myocyte enhancer factor 2 family. MEF2A is widely distributed in various tissues and organs and participates in various physiological processes. This study aimed to investigate the effect of MEF2A expression on the proliferation and apoptosis of bovine myoblasts. CCK8, ELISA, cell cycle, and apoptosis analyses were conducted to assess cell status. In addition, the mRNA expression levels of genes associated with bovine myoblast proliferation and apoptosis were evaluated using RT-qPCR. The results showed that the upregulation of MEF2A mRNA promoted the proliferation rate of myoblasts, shortened the cycle process, and increased the anti-apoptotic rate. Furthermore, the RT-qPCR results showed that the upregulation of MEF2A mRNA significantly increased the cell proliferation factors MyoD1 and IGF1, cell cycle factors CDK2 and CCNA2, and the apoptotic factors Bcl2 and BAD (p < 0.01). These results show that the MEF2A gene can positively regulate myoblast proliferation and anti-apoptosis, providing a basis for the analysis of the regulatory mechanism of the MEF2A gene on bovine growth and development.

E3 ligase Deltex2 accelerates myoblast proliferation and inhibits myoblast differentiation by targeting Pax7 and MyoD, respectively.

E3 ubiquitin ligases are closely related to cell division, differentiation, and survival in all eukaryotes and play crucial regulatory roles in multiple biological processes and diseases. While Deltex2, as a member of the DELTEX family ubiquitin ligases, is characterized by a RING domain followed by a C-terminal domain (DTC), its functions and underlying mechanisms in myogenesis have not been fully elucidated. Here, we report that Deltex2, which is highly expressed in muscles, positively regulates myoblast proliferation via mediating the expression of Pax7. Meanwhile, we find that Deltex2 is translocated from the nucleus into the cytoplasm during myogenic differentiation, and further disclose that Deltex2 inhibits myoblast differentiation and interacts with MyoD, resulting in the ubiquitination and degradation of MyoD. Altogether, our findings reveal the physiological function of Deltex2 in orchestrating myogenesis and delineate the novel role of Deltex2 as a negative regulator of MyoD protein stability.

MiR-708-5p Regulates Myoblast Proliferation and Differentiation.

MicroRNAs (miRNAs) are key regulators involved in the myogenic process in skeletal muscles. miR-708-5p plays an important role in various biochemical and physiological processes, but its function in skeletal myogenesis remain unclear. In this study, we first explored the effects of miR-708-5p on C2C12 proliferation and differentiation by overexpression and interference experiments. Then, we predicted the target genes of miR-708-5p and analyzed their function. We found that miR-708-5p was gradually increased during myoblast differentiation. Overexpression of miR-708-5p significantly inhibited cell proliferation and promoted the differentiation of myoblasts. A total of 253 target genes were predicted using a bioinformatics approach. These genes were significantly enriched in muscle growth-related GO terms and KEGG pathways, such as actin filament organization, actin cytoskeleton organization, PI3K-Akt pathway, insulin pathway, and Jak-STAT pathway. Among them, Pik3ca, Pik3r3, and Irs1 were considered to be the key target genes of miR-708-5p. To sum up, miR-708-5p inhibited C2C12 cells proliferation and promoted C2C12 cells differentiation. Its target genes significantly enriched in GO terms and KEGG pathways related to the development and growth of muscle. Our results provided a basis for studies on the function and mechanism of miR-708-5p regulating skeletal muscle growth and development.

CircCSDE1 Regulates Proliferation and Differentiation of C2C12 Myoblasts by Sponging miR-21-3p.

The growth and development of skeletal muscle is regulated by many factors, and recent studies have shown that circular RNAs (circRNAs) can participate in this process. The model of porcine skeletal muscle injury was constructed to search for circRNAs that can regulate the growth and development of skeletal muscle in pigs. Using whole-transcriptome sequencing and bioinformatics analysis, a novel circRNA (circCSDE1) was screened out, which is highly expressed in skeletal muscle. Functional studies in C2C12 cells demonstrated that circCSDE1 could promote proliferation and inhibit myoblast differentiation, while opposing changes were observed by circCSDE1 knockdown. A dual-luciferase reporter assay revealed that circCSDE1 directly targeted miR-21-3p to regulate the expression of the downstream target gene (Cyclin-dependent kinase 16, CDK16). Moreover, miR-21-3p could inhibit proliferation and promote myoblast differentiation in C2C12 cells, opposite with the effects of circCSDE1. Additionally, the rescue experiments offered further evidence that circCSDE1 and its target, miR-21-3p, work together to regulate myoblast proliferation and differentiation. This study provides a theoretical basis for further understanding the regulatory mechanisms of circRNAs.

Atonal homolog 8/Math6 regulates differentiation and maintenance of skeletal muscle.

Atonal Homolog 8 (Atoh8) belongs to a large superfamily of transcriptional regulators called basic helix-loop-helix (bHLH) transcription factors. Atoh8 (murine homolog “Math6”) has been shown to be involved in organogenesis during murine embryonic development. We have previously identified the expression of Atoh8 during skeletal myogenesis in chicken where we described its involvement in hypaxial myotome formation suggesting a regulatory role of Atoh8 in skeletal muscle development. Within the current study, we analyzed the effect of the loss of function of Atoh8 in murine primary myoblasts and during differentiation of pluripotent stem cells into myotubes, and the effect of its gain of function in C2C12 cells. Based on the observed results, we conclude that Atoh8 regulates myoblast proliferation via modulating myostatin signaling. Further, our data revealed a reduced muscle mass, strength and fiber size with significant changes to the muscle fiber type suggesting atrophy in skeletal muscle of Atoh8 mutants. We further report that Atoh8 knockout mice suffer from a condition similar to ambient hypoxia which may be the primary cause of the phenotype. Altogether, this study shows the significance of Atoh8 not only in myogenesis but also in the maintenance of skeletal muscle.

PHGDH promotes the proliferation and differentiation of primary chicken myoblasts

ABSTRACT 1. Chicken primary myoblasts (CPMs) are precursors that form muscle fibres. The proliferation and differentiation of CPMs is an essential stage in muscle development. Previous RNA-seq analysis showed that phosphoglycerate dehydrogenase (PHGDH) is a differentially expressed gene in chicken muscle tissue at different growth stages. Therefore, the following study explored the effect of PHGDH on the proliferation and differentiation of CPMs. 2. The effect on the proliferation of CPMs by RT–qPCR, CCK-8, and EdU assays after the overexpression and knockdown of PHGDH was evaluated. RT–qPCR, western blotting, and indirect immunofluorescence were used to detect the effect of PHGDH on the differentiation of the CPMs. The expression was observed at different time points for differentiation induced by the CPMs. 3. The results showed that PHGDH significantly promoted proliferation and differentiation in CPMs. The results showed that overexpression of PHGDH significantly upregulated CPM proliferation, while knockdown had the opposite effect. Marker genes showed that overexpression of PHGDH significantly upregulated the expression of P21, MYOG and MYOD genes, significantly downregulated the expression of the MSTN gene and promoted the expression of the MYHC protein. In contrast, PHGDH knockdown had the opposite effect. 4. Desmin immunofluorescence analysis of myotube differentiation in primary myoblasts showed that overexpression of PHGDH significantly increased the area of myotube differentiation and promoted the proliferation and differentiation of myoblasts. Knockdown of PHGDH had the opposite effect. 5. In summary, PHGDH was shown to play a positive role in regulating myoblast proliferation and differentiation. This provides a theoretical basis for further analysis of the regulatory mechanism of the PHGDH gene in chicken muscle development and for improving poultry production.

Dynamic transcriptome profiling reveals essential roles of the Receptor Tyrosine Kinases (RTK) family in feather development of duck

ABSTRACT 1. Chicken primary myoblasts (CPMs) are precursors that form muscle fibres. The proliferation and differentiation of CPMs is an essential stage in muscle development. Previous RNA-seq analysis showed that phosphoglycerate dehydrogenase (PHGDH) is a differentially expressed gene in chicken muscle tissue at different growth stages. Therefore, the following study explored the effect of PHGDH on the proliferation and differentiation of CPMs. 2. The effect on the proliferation of CPMs by RT–qPCR, CCK-8, and EdU assays after the overexpression and knockdown of PHGDH was evaluated. RT–qPCR, western blotting, and indirect immunofluorescence were used to detect the effect of PHGDH on the differentiation of the CPMs. The expression was observed at different time points for differentiation induced by the CPMs. 3. The results showed that PHGDH significantly promoted proliferation and differentiation in CPMs. The results showed that overexpression of PHGDH significantly upregulated CPM proliferation, while knockdown had the opposite effect. Marker genes showed that overexpression of PHGDH significantly upregulated the expression of P21, MYOG and MYOD genes, significantly downregulated the expression of the MSTN gene and promoted the expression of the MYHC protein. In contrast, PHGDH knockdown had the opposite effect. 4. Desmin immunofluorescence analysis of myotube differentiation in primary myoblasts showed that overexpression of PHGDH significantly increased the area of myotube differentiation and promoted the proliferation and differentiation of myoblasts. Knockdown of PHGDH had the opposite effect. 5. In summary, PHGDH was shown to play a positive role in regulating myoblast proliferation and differentiation. This provided a theoretical basis for further analysis of the regulatory mechanism of the PHGDH gene in chicken muscle development and for improving poultry production.

CircRIMKLB promotes myoblast proliferation and inhibits differentiation by sponging miR-29c to release KCNJ12

ABSTRACT Muscle development is a complex process that was regulated by many factors, among which non-coding RNAs (ncRNAs) play a vital role in regulating multiple life activities of muscle cells. Circular RNA (circRNA), a type of non-coding RNA with closed-loop structure, has been reported to affect multiple life processes. However, the roles of circRNAs on muscle development have not been fully elucidated. The present study aimed to determine whether and how circRIMKLB affects muscle development. Our study revealed that circRIMKLB promoted myoblast proliferation and inhibited differentiation. Besides, miR-29c was proved as a downstream target of circRIMKLB using dual-luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay. Also, potassium inwardly rectifying channel subfamily J member 12 (KCNJ12) was identified as a novel target of miR-29c via dual-luciferase reporter assay, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and western blot. CircRIMKLB and KCNJ12 were both proved to regulate cell cycle on muscle regeneration after injury in vivo. In conclusion, we demonstrated that circRIMKLB sponged miR-29c, releasing KCNJ12 to regulate myoblast proliferation and differentiation and regulating cell cycle during muscle regeneration after injury in vivo.

M6A Methylases Regulate Myoblast Proliferation, Apoptosis and Differentiation.

Simple SummaryN6-methyladenosine (m6A) is the most prevalent methylation modification in eukaryotic mRNA, and it plays an important role in regulating gene expression. Previous studies found that m6A methylation plays a role in mammalian skeletal muscle development. Skeletal muscle is an important factor that regulates livestock muscle quality and maintains metabolic homeostasis, and skeletal myogenesis is regulated by a series of transcription factors. However, the role of m6A in bovine skeletal myogenesis is unclear. In this study, we examined the expression patterns of the m6A methylase genes METTL3, METTL14, WTAP, FTO and ALKBH5 in bovine skeletal muscle tissue and during myogenesis in myoblasts. Furthermore, we used bovine skeletal muscle myoblasts as the object of study to discover the regulatory role of these genes in the process of skeletal myogenesis in vitro. Our findings indicate that these five m6A methylases have pronounced and diverse functions in regulating bovine skeletal myoblast proliferation, apoptosis and myogenic differentiation, which can contribute to further understanding the roles of m6A in skeletal muscle development.N6-methyladenosine (m6A) plays an important role in regulating gene expression. Previous studies found that m6A methylation affects skeletal muscle development. However, the effect of m6A methylases on bovine skeletal myogenesis is still unclear. Here, we found that the expression of m6A demethylases (FTO and ALKBH5) was significantly higher in the longissimus dorsi muscle of adult cattle than in newborn cattle. In contrast, the expression of m6A methyltransferases (METTL3, METTL14 and WTAP) was reduced. The mRNA expression of all five genes was found to be increased during the myogenesis of myoblasts in vitro. Knockdown of FTO or METTL3 promoted myoblast proliferation, inhibited myoblast apoptosis and suppressed myogenic differentiation, whereas ALKBH5 knockdown had the opposite effect. METTL14 knockdown enhanced myoblast proliferation and impaired myogenic differentiation. WTAP knockdown attenuated proliferation and contributed to apoptosis but did not affect differentiation. Furthermore, the functional domains of these five m6A methylases are conserved across species. Our results suggest that m6A methylases are involved in regulating skeletal muscle development and that there may be a complex network of m6A methylation regulating skeletal myogenesis.

LncEDCH1 improves mitochondrial function to reduce muscle atrophy by interacting with SERCA2

Skeletal muscle is a regulator of the body's energy expenditure and metabolism. Abnormal regulation of skeletal muscle-specific genes leads to various muscle diseases. Long non-coding RNAs (lncRNAs) have been demonstrated to play important roles in muscle growth and muscle atrophy. To explore the potential function of muscle-associated lncRNA, we analyzed our previous RNA-sequencing data and selected the lncRNA (LncEDCH1) as the research object. In this study, we report that LncEDCH1 is specifically enriched in skeletal muscle, and its transcriptional activity is positively regulated by transcription factor SP1. LncEDCH1 regulates myoblast proliferation and differentiation in vitro. In vivo, LncEDCH1 reduces intramuscular fat deposition, activates slow-twitch muscle phenotype, and inhibits muscle atrophy. Mechanistically, LncEDCH1 binds to sarcoplasmic/ER calcium ATPase 2 (SERCA2) protein to enhance SERCA2 protein stability and increase SERCA2 activity. Meanwhile, LncEDCH1 improves mitochondrial efficiency possibly through a SERCA2-mediated activation of the AMPK pathway. Our findings provide a strategy for using LncEDCH1 as an effective regulator for the treatment of muscle atrophy and energy metabolism.

Effects of hyperoxia and hypoxia on the proliferation of C2C12 myoblasts.

Hyperoxic conditions are known to accelerate skeletal muscle regeneration after injuries. In the early phase of regeneration, macrophages invade the injured area and subsequently secrete various growth factors, which regulate myoblast proliferation and differentiation. Although hyperoxic conditions accelerate muscle regeneration, it is unknown whether this effect is indirectly mediated by macrophages. Here, using C2C12 cells, we show that not only hyperoxia but also hypoxia enhance myoblast proliferation directly, without accelerating differentiation into myotubes. Under hyperoxic conditions (95% O2 + 5% CO2), the cell membrane was damaged because of lipid oxidization, and a disrupted cytoskeletal structure, resulting in suppressed cell proliferation. However, a culture medium containing vitamin C (VC), an antioxidant, prevented this lipid oxidization and cytoskeletal disruption, resulting in enhanced proliferation in response to hyperoxia exposure of ≤4 h/day. In contrast, exposure to hypoxic conditions (95% N2 + 5% CO2) for ≤8 h/day enhanced cell proliferation. Hyperoxia did not promote cell differentiation into myotubes, regardless of whether the culture medium contained VC. Similarly, hypoxia did not accelerate cell differentiation. These results suggest that regardless of hyperoxia or hypoxia, changes in oxygen tension can enhance cell proliferation directly, but do not influence differentiation efficiency in C2C12 cells. Moreover, excess oxidative stress abrogated the enhancement of myoblast proliferation induced by hyperoxia. This research will contribute to basic data for applying the effects of hyperoxia or hypoxia to muscle regeneration therapy.

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

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.

FTO regulates myoblast proliferation by controlling CCND1 expression in an m6A-YTHDF2-dependent manner

N6-Methyladenosine (m6A) modification is the most abundant chemical modification in mRNA, and it participates in various biological processes, such as cell differentiation and proliferation. However, little is known about the function of m6A demethylase fat mass and obesity-associated (FTO) in myoblast proliferation. Here, we demonstrated that knockdown of FTO can significantly inhibit myoblast proliferation and promote apoptosis. RNA sequencing analysis revealed that a lot of downregulated genes in FTO knockdown cells are associated with cell cycle and apoptosis. Furthermore, silencing FTO drastically decreased cyclin D1 (CCND1) expression through YTHDF2-mediated mRNA degradation, thereby delaying the progression of G1 phase, and leading to impaired myoblast proliferation. These findings unraveled that FTO regulates myoblast proliferation by controlling CCND1 expression in an m6A-YTHDF2-dependent manner, which highlights the critical roles of m6A modification in myoblast proliferation.