Skeletal Muscle Cell Proliferation Research Articles

An intronic SNP affects skeletal muscle development by regulating the expression of TP63.

Porcine skeletal muscle development is pivotal for improving meat production. TP63, a transcription factor, regulates vital cellular processes, yet its role in skeletal muscle proliferation is unclear. The effects of TP63 on skeletal muscle cell viability and proliferation were investigated using both mouse and porcine skeletal muscle myoblasts. Selective sweep analysis in Western pigs identified TP63 as a potential candidate gene for skeletal muscle development. The correlation between TP63 overexpression and cell proliferation was assessed using quantitative real-time PCR (RT-qPCR) and 5-ethynyl-2'-deoxyuridine (EDU). The study revealed a positive correlation between TP63 overexpression and skeletal muscle cell proliferation. Bioinformatics analysis predicted an interaction between MEF2A, another transcription factor, and the mutation site of TP63. Experimental validation through dual-luciferase assays confirmed that a candidate enhancer SNP could influence MEF2A binding, subsequently regulating TP63 expression and promoting skeletal muscle cell proliferation. These findings offer experimental evidence for further exploration of skeletal muscle development mechanisms and the advancement of genetic breeding strategies aimed at improving meat production traits.

Overexpression of GPX2 gene regulates the development of porcine preadipocytes and skeletal muscle cells through MAPK signaling pathway.

Glutathione peroxidase 2 (GPX2) is a selenium-dependent enzyme and protects cells against oxidative damage. Recently, GPX2 has been identified as a candidate gene for backfat and feed efficiency in pigs. However, it is unclear whether GPX2 regulates the development of porcine preadipocytes and skeletal muscle cells. In this study, adenoviral gene transfer was used to overexpress GPX2. Our findings suggest that overexpression of GPX2 gene inhibited proliferation of porcine preadipocytes. And the process is accompanied by the reduction of the p-p38. GPX2 inhibited adipogenic differentiation and promoted lipid degradation, while ERK1/2 was reduced and p-p38 was increased. Proliferation of porcine skeletal muscle cells was induced after GPX2 overexpression, was accompanied by activation in JNK, ERK1/2, and p-p38. Overexpression methods confirmed that GPX2 has a promoting function in myoblastic differentiation. ERK1/2 pathway was activated and p38 was suppressed during the process. This study lays a foundation for the functional study of GPX2 and provides theoretical support for promoting subcutaneous fat reduction and muscle growth.

SOX6 AU controls myogenesis by cis-modulation of SOX6 in cattle

ABSTRACT Long non-coding RNAs (lncRNAs) have been shown to be involved in the regulation of skeletal muscle development through multiple mechanisms. The present study revealed that the lncRNA SOX6 AU (SRY-box transcription factor 6 antisense upstream) is reverse transcribed from upstream of the bovine sex-determining region Y (SRY)-related high-mobility-group box 6 (SOX6) gene. SOX6 AU was significantly differentially expressed in muscle tissue among different developmental stages in Xianan cattle. Subsequently, knockdown and overexpression experiments discovered that SOX6 AU promoted primary skeletal muscle cells proliferation, apoptosis, and differentiation in bovine. The overexpression of SOX6 AU in bovine primary skeletal muscle cells resulted in 483 differentially expressed genes (DEGs), including 224 upregulated DEGs and 259 downregulated DEGs. GO functional annotation analysis showed that muscle development-related biological processes such as muscle structure development and muscle cell proliferation were significantly enriched. KEGG pathway analysis revealed that the PI3K/AKT and MAPK signaling pathways were important pathways for DEG enrichment. Notably, we found that SOX6 AU inhibited the mRNA and protein expression levels of the SOX6 gene. Moreover, knockdown of the SOX6 gene promoted the proliferation and apoptosis of bovine primary skeletal muscle cells. Finally, we showed that SOX6 AU promoted the proliferation and apoptosis of bovine primary skeletal muscle cells by cis-modulation of SOX6 in cattle. This work illustrates our discovery of the molecular mechanisms underlying the regulation of SOX6 AU in the development of beef.

Myokines Produced by Cultured Bovine Satellite Cells Harvested from 3- and 11-Month-Old Angus Steers.

The myokines interleukin 6 (IL-6), interleukin 15 (IL-15), myonectin (CTRP15), fibronectin type III domain containing protein 5/irisin (FNDC5), and brain-derived neurotrophic factor (BDNF) are associated with skeletal muscle cell proliferation, differentiation, and muscle hypertrophy in biomedical model species. This study evaluated whether these myokines are produced by cultured bovine satellite cells (BSCs) harvested from 3- and 11-month-old commercial black Angus steers and if the expression and secretion of these targets change across 0, 12, 24, and 48 h in vitro. IL-6, IL-15, FNDC5, and BDNF expression were greater (p ≤ 0.05) in the differentiated vs. undifferentiated BSCs at 0, 12, 24, and 48 h. CTRP15 expression was greater (p ≤ 0.03) in the undifferentiated vs. differentiated BSCs at 24 and 48 h. IL-6 and CTRP15 protein from culture media were greater (p ≤ 0.04) in undifferentiated vs. differentiated BSCs at 0, 12, 24, and 48 h. BDNF protein was greater in the media of differentiated vs. undifferentiated BSCs at 0, 12, 24, and 48 h. IL-6, 1L-15, FNDC5, and BDNF are expressed in association with BSC differentiation, and CTRP15 appears to be expressed in association with BSC proliferation. This study also confirms IL-6, IL-15, CTRP15, and BDNF proteins present in media collected from primary cultures of BSCs.

Transcription Factor SATB2 Regulates Skeletal Muscle Cell Proliferation and Migration via HDAC4 in Pigs.

Skeletal muscle development remarkably affects meat production and growth rate, regulated by complex regulatory mechanisms in pigs. Specific AT sequence-binding protein 2 (SATB2) is a classic transcription factor and chromatin organizer, which holds a profound effect in the regulation of chromatin remodeling. However, the regulation role of SATB2 concerning skeletal muscle cell fate through chromatin remodeling in pigs remains largely unknown. Here, we observed that SATB2 was expressed higher in the lean-type compared to the obese-type pigs, which also enriched the pathways of skeletal muscle development, chromatin organization, and histone modification. Functionally, knockdown SATB2 led to decreases in the proliferation and migration markers at the mRNA and protein expression levels, respectively, while overexpression SATB2 had the opposite effects. Further, we found histone deacetylase 4 (HDAC4) was a key downstream target gene of SATB2 related to chromatin remodeling. The binding relationship between SATB2 and HDAC4 was confirmed by a dual-luciferase reporter system and ChIP-qPCR analysis. Besides, we revealed that HDAC4 promoted the skeletal muscle cell proliferation and migration at the mRNA and protein expression levels, respectively. In conclusion, our study indicates that transcription factor SATB2 binding to HDAC4 positively contributes to skeletal muscle cell proliferation and migration, which might mediate the chromatin remodeling to influence myogenesis in pigs. This study develops a novel insight into understanding the molecular regulatory mechanism of myogenesis, and provides a promising gene for genetic breeding in pigs.

Deciphering the population structure and genetic basis of growth traits from whole-genome resequencing of the leopard coral grouper ( Plectropomus leopardus).

The leopard coral grouper ( Plectropomus leopardus) is a species of significant economic importance. Although artificial cultivation of P. leopardus has thrived in recent decades, the advancement of selective breeding has been hindered by the lack of comprehensive population genomic data. In this study, we identified over 8.73 million single nucleotide polymorphisms (SNPs) through whole-genome resequencing of 326 individuals spanning six distinct groups. Furthermore, we categorized 226 individuals with high-coverage sequencing depth (≥14×) into eight clusters based on their genetic profiles and phylogenetic relationships. Notably, four of these clusters exhibited pronounced genetic differentiation compared with the other populations. To identify potentially advantageous loci for P. leopardus, we examined genomic regions exhibiting selective sweeps by analyzing the nucleotide diversity ( θπ) and fixation index ( F ST) in these four clusters. Using these high-coverage resequencing data, we successfully constructed the first haplotype reference panel specific to P. leopardus. This achievement holds promise for enabling high-quality, cost-effective imputation methods. Additionally, we combined low-coverage sequencing data with imputation techniques for a genome-wide association study, aiming to identify candidate SNP loci and genes associated with growth traits. A significant concentration of these genes was observed on chromosome 17, which is primarily involved in skeletal muscle and embryonic development and cell proliferation. Notably, our detailed investigation of growth-related SNPs across the eight clusters revealed that cluster 5 harbored the most promising candidate SNPs, showing potential for genetic selective breeding efforts. These findings provide a robust toolkit and valuable insights into the management of germplasm resources and genome-driven breeding initiatives targeting P. leopardus.

The YBX3 RNA-binding protein posttranscriptionally controls SLC1A5 mRNA in proliferating and differentiating skeletal muscle cells

In humans, skeletal muscles comprise nearly 40% of total body mass, which is maintained throughout adulthood by a balance of muscle protein synthesis and breakdown. Cellular amino acid (AA) levels are critical for these processes, and mammalian cells contain transporter proteins that import AAs to maintain homeostasis. Until recently, the control of transporter regulation has largely been studied at the transcriptional and post-translational levels. However, here we report that the RNA-binding protein YBX3 is critical to sustain intracellular AAs in mouse skeletal muscle cells, which aligns with our recent findings in human cells. We find that YBX3 directly binds the SLC1A5 AA transporter messenger (m)RNA to post-transcriptionally control SLC1A5 expression during skeletal muscle cell differentiation. YBX3 regulation of SLC1A5 requires the 3’ untranslated region (UTR). Additionally, intracellular AAs transported by SLC1A5, either directly or indirectly through coupling to other transporters, are specifically reduced when YBX3 is depleted. Further, we find that reduction of the YBX3 protein reduces proliferation and impairs differentiation in skeletal muscle cells, and that YBX3 and SLC1A5 protein expression increase substantially during skeletal muscle differentiation, independently of their respective mRNA levels. Taken together, our findings suggest that YBX3 regulates AA transport in skeletal muscle cells and that its expression is critical to maintain skeletal muscle cell proliferation and differentiation.

Moracin E and M isolated from Morus alba Linné induced the skeletal muscle cell proliferation via PI3K-Akt-mTOR signaling pathway

Twigs of Morus alba have been used in traditional medicine to treat muscle-related symptoms such as aches, numbness, and stiffness. Despite its clinical use in traditional medicine, its active compounds and mode of action have not yet been investigated. Therefore, we aimed to isolate the compounds from the twigs of M. alba and deduce active compounds, key gene targets, and mechanism of action against sarcopenia using network pharmacology analysis. Using various isolation techniques and spectroscopic methods, 43 phytochemicals, including 3 new flavonoids, were isolated and performed network pharmacology analysis. According to the computational-assistant analysis, 28 compounds, 9 genes, and the PI3K-Akt-mTOR signaling pathway were deduced as expected active compounds (EAC), key targets, and the main signaling pathway. To verify the predicted results, the cell proliferation activities of the EAC were evaluated. Especially, moracin E and M significantly increased by 130% (p < 0.001) and 57% (p < 0.05), respectively, which have more than 2- and 1.5-fold stronger effects compared to the control. Furthermore, both increased the expression level of proteins involved in the PI3K-Akt-mTOR signaling pathway and myogenic proteins, including myogenin and MyoD. This study demonstrated that moracin E and M exhibit cell proliferative effects on skeletal muscle cells through the PI3K-Akt-mTOR signaling pathway.

A newly identified lncRNA lnc000100 regulates proliferation and differentiation of cattle skeletal muscle cells

ABSTRACT Cattle skeletal muscle development is a complex and highly coordinated biological process mediated by a series of myogenic regulators, which plays a critical role in beef yield and quality. Long non-coding RNAs (lncRNAs) have been shown to regulate skeletal muscle development. However, the molecular mechanism by which lncRNAs regulate skeletal muscle development is largely unknown. We performed transcriptome analysis of muscle tissues of adult and embryo Angus cattle to investigate the mechanism by which lncRNA regulates skeletal muscle development between adult and embryo cattle. A total of 37,115 candidate lncRNAs were detected, and a total of 1,998 lncRNAs were differentially expressed between the muscle tissue libraries of adult and embryo cattle, including 1,229 up-regulated lncRNAs and 769 down-regulated lncRNAs (adult cattle were the control group). We verified the expression of 7 differentially expressed lncRNAs by quantitative real-time PCR (RT-qPCR), and analysed the tissue expression profile of lnc000100, which is down-regulated in the longest dorsal muscle during foetal life and which is highly specifically expressed in muscle tissue. We found that the interference of lnc000100 significantly inhibited cell proliferation and promoted cell differentiation. Lnc000100 was located in the nucleus by RNA-FISH. Our research provides certain resources for the analysis of lncRNA regulating cattle skeletal muscle development, and may also provide new insights for improving beef production and breed selection.

Tissue regeneration and gut microbiota-skeletal muscle axis via microRNAs and nutrition: a systematic review

Introduction: In the scenario of muscle regeneration in athletes, maintenance of skeletal muscle function is the prerequisite for tissue homeostasis and increased performance. MicroRNAs play a positive role in expanding our understanding of the controlling factors for skeletal muscle function. Recent progress has been made regarding gut microbiota, regenerative nutrition, and skeletal muscle metabolism. Objective: It was to carry out a systematic review of the main relations of regeneration of skeletal muscle-gut microbiota through nutrological functions, cells, and microRNAs. Methods: The systematic review rules of the PRISMA Platform were followed. The research was carried out from February to April 2023 in Scopus, PubMed, Science Direct, Scielo, and Google Scholar databases. The quality of the studies was based on the GRADE instrument and the risk of bias was analyzed according to the Cochrane instrument. Results and Conclusion: A total of 217 articles were found. A total of 97 articles were fully evaluated and 49 were included and developed in the present systematic review study. Considering the Cochrane tool for risk of bias, the overall assessment resulted in 54 studies at high risk of bias and 34 studies that did not meet the GRADE. It was concluded that miRNAs are widely present in skeletal muscle and play an irreplaceable tuning role in the proliferation, differentiation, apoptosis, development, and other physiological processes of skeletal muscle cells. The proposal that miRNAs are primarily involved in the cell's stress response makes miRNAs ideal for mediating the skeletal muscle response to changes in contractile activity. Research has accumulated evidence that confirms that miRNAs have played an important regulatory role in cell proliferation and differentiation, thus regulating skeletal muscle growth as highlighted in the small intestine by intestinal stem cells (LGR5+). The ketogenic or high-glucose diet regulates the self-renewal balance of LGR5+. Self-renewal and HSC differentiation can be regulated by manipulating vitamin C, A, or D levels and valine restriction. The composition of each athlete's microbiome influences sports performance.

Four new lignans obtained from the leaves of Schisandra cauliflora and their effect on skeletal muscle cell proliferation.

Plants of the Schisandra genus are commonly used in folk medicinal remedies. Some Schisandra species and their lignans have been reported to improve muscle strength. In the present study, four new lignans, named schisacaulins A-D, together with three previously described compounds ananonin B, alismoxide, and pregomisin were isolated from the leaves of S. cauliflora. Their chemical structures were determined by extensive analyses of HR-ESI-MS, NMR, and ECD spectra. Schisacaulin D and alismoxide significantly stimulated skeletal muscle cell proliferation by increasing the number of fused myotubes and expression of myosin heavy chain (MyHC) which may be good candidates for the treatment of sarcopenia.

The VGLL2 gene participates in muscle development in Gushi chickens1

Previous studies have shown that VGLL2, a member of the VGLL family, plays important roles in the growth and development of animal skeletal muscle, but its specific role in the development of chicken skeletal muscle is unclear. The main goal of this study was to explore the biological functions of VGLL2 in the development of chicken skeletal muscle and the proliferation and differentiation of skeletal muscle cells in vitro. In this study, we detected the effect of VGLL2 on the proliferation of myoblasts by CCK8, EdU and flow cytometry analyses after overexpressing and interfering with VGLL2. Indirect immunofluorescence was used to detect the effect of VGLL2 on the differentiation of myoblasts. qRT-PCR and hematoxylin and eosin (H&E) staining were used to evaluate the effects of VGLL2 overexpression on the growth rate and muscle fiber structure of chicken skeletal muscle. The results showed that VGLL2 inhibited the proliferation of primary cultured chicken myoblasts and promoted the differentiation of these cells. Interestingly, food intake and muscle fiber development were significantly enhanced by the overexpression of VGLL2 in chickens. Taken together, these data demonstrate that the VGLL2 gene may be a useful marker for improving muscle mass in poultry.

DEHP exposure impairs human skeletal muscle cell proliferation in primary culture conditions: preliminary study.

DEHP exposure impairs human skeletal muscle cell proliferation in primary culture conditions: preliminary study.

Discovery of 2 New Dibenzocyclooctadiene Lignans Cauliflorins A and B From Schisandra cauliflora

Sarcopenia is having a serious impact on highly aged populations. A Japanese traditional medicinal remedy containing Schisandra fruits and Astragalus roots recently showed effective treatment of sarcopenia and thus needed to be investigated further. This current study selected Schisandra cauliflora fruits for investigation of their chemical constituents and evaluation of their antisarcopenia effects. Dried S cauliflora fruits were ultrasonically extracted with methylene chloride. This extract was purified by a combination of chromatographic techniques. The isolated compounds were characterized by nuclear magnetic resonance spectroscopy, electronic circular dichroism, and high resolution electrospray ionization mass spectrometry methods. Antisarcopenia activities of the isolated compounds were evaluated by their effects on the viability and proliferation of C2C12 skeletal muscle cells. Seven dibenzocyclooctadiene lignans, including 2 that had been previously undescribed, cauliflorins A and B (1 and 2), ananolignan C (3), 6- epi-ananolignan D (4), ananolignan F (5), ananonin H (6), and ananonin J (7) were isolated from the methylene chloride extract of the fruits of S cauliflora. The C2C12 cells treated with compounds 1-7 (5 µM) exhibited proliferation in the range of 75.7% ± 7.4% to 98.2% ± 11.7%, compared to 100.0% ± 6.0% of the untreated cells. Two previously undescribed and 5 known dibenzocyclooctadiene lignans were isolated from S cauliflora fruits. All the isolated compounds were inactive on the proliferative enhancement of C2C12 skeletal muscle cells.

Biological functions of circRNAs and their advance on skeletal muscle development in bovine.

The development of skeletal muscle in animals is a complex biological process, which are strictly and precisely regulated by many genes and non-coding RNAs. Circular RNA (circRNA) was found as a novel class of functional non-coding RNA with ring structure in recent years, which appears in the process of transcription and is formed by covalent binding of single-stranded RNA molecules. With the development of sequencing and bioinformatics analysis technology, the functions and regulation mechanisms of circRNAs have attracted great attention due to its high stability characteristics. The role of circRNAs in skeletal muscle development have been gradually revealed, where circRNAs were involved in various biological processes, such as proliferation, differentiation, and apoptosis of skeletal muscle cells. In this review, we summarized the current studies advance of circRNAs involved in skeletal muscle development in bovine, and hope to gain a deeper understanding of the functional roles of the circRNAs in muscle growth. Our results will provide some theoretical supports and great helps for the genetic breeding of this species, and aiming at improving bovine growth and development and preventing muscle diseases.

Bioactive cellulose acetate nanofiber loaded with annatto support skeletal muscle cell attachment and proliferation.

Electrospinning emerged as a promising technique to produce scaffolds for cultivated meat in function of its simplicity, versatility, cost-effectiveness, and scalability. Cellulose acetate (CA) is a biocompatible and low-cost material that support cell adhesion and proliferation. Here we investigated CA nanofibers, associated or not with a bioactive annatto extract (CA@A), a food-dye, as potential scaffolds for cultivated meat and muscle tissue engineering. The obtained CA nanofibers were evaluated concerning its physicochemical, morphological, mechanical and biological traits. UV-vis spectroscopy and contact angle measurements confirmed the annatto extract incorporation into the CA nanofibers and the surface wettability of both scaffolds, respectively. SEM images revealed that the scaffolds are porous, containing fibers with no specific alignment. Compared with the pure CA nanofibers, CA@A nanofibers showed increased fiber diameter (420 ± 212nm vs. 284 ± 130nm). Mechanical properties revealed that the annatto extract induces a reduction of the stiffness of the scaffold. Molecular analyses revealed that while CA scaffold favored C2C12 myoblast differentiation, the annatto-loaded CA scaffold favored a proliferative state of these cells. These results suggest that the combination of cellulose acetate fibers loaded with annatto extract may be an interesting economical alternative for support long-term muscle cells culture with potential application as scaffold for cultivated meat and muscle tissue engineering.

The identification and validation of target genes of IGFBP3 protein in sheep skeletal muscle cells

This study aimed to identify the target genes of IGFBP3（insulin growth factor binding protein）protein and to investigate its target genes effects on the proliferation and differentiation of Hu sheep skeletal muscle cells. IGFBP3 was an RNA-binding protein that regulates mRNA stability. Previous studies have reported that IGFBP3 promotes the proliferation of Hu sheep skeletal muscle cells and inhibits differentiation, but the downstream genes that bind to it have not been reported yet. We predicted the target genes of IGFBP3 through RNAct and sequencing data, and verified by qPCR and RIP（RNA Immunoprecipitation）experiments, and demonstrated GNAI2（G protein subunit alpha i2）as one of the target gene of IGFBP3. After interference with siRNA, we carried out qPCR, CCK8, EdU, and immunofluorescence experiments, and found that GNAI2 can promote the proliferation and inhibit differentiation of Hu sheep skeletal muscle cells. This study revealed the effects of GNAI2 and provided one of the regulatory mechanisms of IGFBP3 protein underlying sheep muscle development.

Mt-Ty 5'tiRNA regulates skeletal muscle cell proliferation and differentiation.

In this study, we sought to determine the role of tRNA-derived fragments in the regulation of gene expression during skeletal muscle cell proliferation and differentiation. We employed cell culture to examine the function of mt-Ty 5' tiRNAs. Northern blotting, RT-PCR as well as RNA-Seq, were performed to determine the effects of mt-Ty 5' tiRNA loss and gain on gene expression. Standard and transmission electron microscopy (TEM) were used to characterize cell and sub-cellular structures. mt-Ty 5'tiRNAs were found to be enriched in mouse skeletal muscle, showing increased levels in later developmental stages. Gapmer-mediated inhibition of tiRNAs in skeletal muscle C2C12 myoblasts resulted in decreased cell proliferation and myogenic differentiation; consistent with this observation, RNA-Seq, transcriptome analyses, and RT-PCR revealed that skeletal muscle cell differentiation and cell proliferation pathways were also downregulated. Conversely, overexpression of mt-Ty 5'tiRNAs in C2C12 cells led to a reversal of these transcriptional trends. These data reveal that mt-Ty 5'tiRNAs are enriched in skeletal muscle and play an important role in myoblast proliferation and differentiation. Our study also highlights the potential for the development of tiRNAs as novel therapeutic targets for muscle-related diseases.

Porcine IGF-1R synonymous mutations in the extracellular domain affect proliferation and differentiation of skeletal muscle cells

ObjectiveMiniature pigs are considered ideal organ donors for xenotransplantation in humans, but the mechanism underlying their dwarfism remains to be elucidated. IGF-1R is a crucial factor in body size formation in mammals, including skeletal muscle formation and development. The extracellular domain (ECD) binds to the ligand, a phenomenon that results in the activation of downstream pathways. MethodsIn this study, the coding sequences of two IGF-1R ECD haplotypes of the large Landrace (LP) pig and the small Bama Xiang (BM) pig were cloned into pcDNA3.1 vectors to generate pcDNA3.1-LP and pcDNA3.1-BM. The two recombinant vectors were then transfected into skeletal muscle cells. ResultsIGF-1R transcript was found to be expressed at higher levels in the pcDNA3.1-LP group than in the pcDNA3.1-BM group. The IGF-1R ECD from LP promoted cell proliferation and CyclinD1 expression, and promoted the phosphorylation of protein kinase B (to yield p-AKT). Moreover, the IGF-1R ECD from LP increased cell differentiation and the expression of myogenic determination factor (MyoD). ConclusionOur data indicated that the IGF-1R ECD haplotypes between pig breeds with different body sizes affect IGF-1R expression, in turn affecting the proliferation and differentiation of skeletal muscle cells by activating downstream signalling pathways.

Transcriptome analysis of the inhibitory effect of cycloleucine on myogenesis

N6-Methyladenosine (m6A) has been reported to involve and play an important role in various biological activities but seldom in poultry myogenesis. Cycloleucine usually functions as a nucleic acid methylation inhibitor, the inhibition efficiency of cycloleucine at the m6A level and corresponding dynamic changes of poultry muscle cells remain unknown. In this study, we aim to find out the effect of cycloleucine on the total N6-Methyladenosine level and its molecular mechanism for regulating myogenesis. A total of 745 differentially expressed genes (DEGs) were obtained by 10 mM, 20 mM, and 30 mM of cycloleucine treatment compared with 0 mM treatment. DEGs in 10 mM cycloleucine were significantly enriched in the biological process of skeletal muscle and satellite cell proliferation and differentiation, DEGs in 20 and 30 mM cycloleucine were enriched in some metabolic and biosynthetic processes. The trend analysis showed that 85% of all DEGs were significantly clustered into 4 files, among them 59% DEGs were dose-dependent and 26% were dose-independent, 52% DEGs were in downtrend and 33% DEGs were in uptrend. Also, the cycloleucine treatment could trigger cell cycle arrest in the G1 phase and depress myoblast cell proliferation and inhibit myotube formation. In conclusion, cycloleucine could continuously reduce the m6A level of myoblast cells, depress myoblast cell proliferation and inhibit myotube formation.