Chicken Myoblasts Research Articles

Use of transcriptome sequencing to explore the effect of CSRP3 on chicken myoblasts

The mechanisms that regulate the specificity and maintenance of chicken muscle fiber types remain largely unknown. In mammals, CSRP3 has been shown to play a vital role in the maintenance of typical muscle structure and function. This study investigated the role that CSRP3 plays in chicken skeletal muscle. First, the antibody against chicken CSRP3 protein was prepared, and the expression levels of the mRNA and protein of the CSRP3 gene in four chicken skeletal muscles with different myofiber compositions were compared. Then the effects of CSRP3 silencing on the expression profile of chicken myoblast transcriptomes were analyzed. The results showed that the expression levels of the mRNA and protein of the CSRP3 gene were both associated with the composition of fiber types in chicken skeletal muscles. A total of 650 genes with at least 1.5-fold differences (Q<0.05) were identified, of which 255 genes were upregulated and 395 genes were downregulated by CSRP3 silencing. Functional enrichment showed that several pathways, including adrenergic signaling in cardiomyocytes, adipocytokine signaling pathway and apelin signaling pathway, were significantly (P<0.05) enriched both in differentially expressed genes and all expressed genes. The co-expressed gene network suggested that CSRP3 silencing caused a compensatory upregulation (Q<0.05) of genes related to the assembly of myofibrils, muscle differentiation, and contraction. Meanwhile, two fast myosin heavy chain genes (MyH1B and MyH1E) were upregulated (Q<0.05) upon CSRP3 silencing. These results suggested that CSRP3 plays a crucial role in chicken myofiber composition, and affects the distribution of chicken myofiber types, probably by regulating the expression of MyH1B and MyH1E.

MiR-181a-5p can inhibit the proliferation and promote the differentiation of chicken primary myoblasts

ABSTRACT 1. Myoblast proliferation and differentiation is one of the most important biological processes in the development of skeletal muscle. MicroRNAs (miRNAs) play a crucial role in this process. 2. In this study, the expression level of miR-181a-5p was detected, which found that miR-181a-5p was expressed differently in different tissues, different embryonic ages, and different differentiation stages of primary myoblasts in Gushi chickens. 3. The effect of miR-181a-5p was further investigated on chicken primary myoblasts (CPMs). The results of cell counting kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU) and cell cycle showed that miR-181a-5p could inhibit the proliferation of CPM. The miR-181a-5p promoted the expression of MYOD, MYOG, and MYHC. MYHC protein immunofluorescence experiments showed that miR-181a-5p increased the area of myotubes. 4. In total, 63 potential target genes of miR-181a-5p in mRNA transcriptome data analysis were identified. Functional enrichment analysis was performed on these target genes, and ASNS, SMYD1, and FOS were found to play regulatory roles in biological processes such as muscle development. It was speculated that miR-181a-5p played a role in myoblast development through these genes. 5. In conclusion, miR-181a-5p can inhibit the proliferation of chicken myoblasts and promote the differentiation of chicken myoblasts. This study laid the foundation for further research on the regulatory mechanism of miR-181a-5p in the development of skeletal muscle and the formation of excellent meat quality traits in Gushi chicken.

Adiponectin Reduces Lipid Content in Chicken Myoblasts by Activating AMPK Signaling Pathway.

Studies in mammals have shown that adiponectin is secreted mainly by adipocytes, and it plays a crucial role in glucose and lipid metabolism in muscles. Clarifying the cross-talk role of adiponectin between adipose tissue and skeletal muscle tissue is very important for internal homeostasis. The glucose and lipid metabolism of chicken is different from that of mammals, and the role of adiponectin in chickens is unclear. Therefore, it is of great significance to study the effect and mechanism of adiponectin on lipid metabolism in chickens. In the present study, the regulating effect of adiponectin on lipid metabolism in chicken myoblasts was explored by adding a certain concentration of exogenous recombinant adiponectin. Results showed that adiponectin reduced intracellular lipid content, increasing the mRNA expression of adiponectin receptor and cellular uptake of glucose and fatty acids. In addition, adiponectin activated the 5′ adenosine monophosphate activated protein kinase (AMPK) signaling pathway. The above results suggested that adiponectin reduced intracellular lipid content, mainly by binding to adiponectin receptor, activating AMPK pathway, increasing cellular uptake of glucose and fatty acids and promoting lipid oxidation.

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.

C-type natriuretic peptide stimulates chicken myoblast differentiation through NPRB/NPRC receptors and metabolism pathway

Skeletal muscle development is closely related with the amount of meat production and its quality in chickens. Natriuretic peptides (NPs) play an important role in myotube formation and fat oxidation of skeletal muscle in animals. The effect of C-type natriuretic peptide (CNP), an important member of the NPs, and its underlying molecular mechanisms in skeletal muscle are incompletely understood. Treatment of myoblasts with CNP led to enhanced proliferation/differentiation and significantly upregulated (P<0.05) mRNA expression of the CNP receptors natriuretic peptide receptor B (NPRB) and the clearance receptor C (NPRC). In cells exposed to CNP, 142 differentially expressed genes (84 up-regulation and 58 down-regulation) (P<0.05) were identified by RNA-sequencing compared with those in control cells. Sixteen genes were significantly enriched (P<0.05) in the metabolic pathway, and six of them (phospholipase C β4, phospholipase C β2, phosphoglycerate mutase 1, creatine kinase B, peroxiredoxin 6 and CD38) were closely related to skeletal muscle development and differentially expressed. In conclusion, CNP stimulated differentiation of myoblasts by upregulating expression of the NPRB and NPRC receptors and enriching key genes in the metabolic pathway.

ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion.

SummaryMyoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes. CaMKII activation results in myotube growth via fusion with mononucleated myoblasts at a fusogenic synapse. Mechanistically, CaMKII interacts with and regulates MYMK and Rac1, and CaMKIIδ/γ knockout mice exhibit smaller regenerated myofibers following injury. In addition, the expression of a dominant negative CaMKII inhibits the formation of large multinucleated myotubes. Finally, we demonstrate the evolutionary conservation of the pathway in chicken myoblasts. We conclude that ERK1/2 represses a signaling cascade leading to CaMKII-mediated fusion of myoblasts to myotubes, providing an attractive target for the cultivated meat industry and regenerative medicine.

MiR-29b-1-5p regulates the proliferation and differentiation of chicken primary myoblasts and analysis of its effective targets

Several recent studies investigated the role of the miR-29 family in muscle development. However, only a few studies focused on chicken skeletal muscle. In the present study, cell cycle, 5-ethynyl-2’-deoxyuridine (EdU), cell counting kit-8 (CCK-8), and other assays indicated that miR-29b-1-5p can inhibit the proliferation of chicken primary myoblasts (CPMs); the western blot assay and immunofluorescence detection of MYHC demonstrated that miR-29b-1-5p can promote the differentiation of myoblasts. The functional enrichment analysis revealed that the target genes of miR-29b-1-5p may be involved in muscle tissue development, muscle organ development, and striated muscle tissue development, which are biological processes related to muscle development. The correlation analysis showed that these 6 genes, that is, ankyrin repeat domain 9 (ANKRD9), lactate dehydrogenase A (LDHA), transcription factor 12 (TCF12), FAT atypical cadherin 1 (FAT1), lin-9 homolog (LIN9), and integrin beta 3 binding protein (ITGB3BP), can be used as effective candidate target genes of miR-29b-1-5p. Moreover, miR-29b-1-5p inhibits the expression of ANKRD9 by directly binding the 3′UTR of ANKRD9. Overall, these data indicate that miR-29b-1-5p inhibits the proliferation of primary chicken myoblasts, stimulates their differentiation, and is involved in the process of muscle development and that its effective target gene is ANKRD9. This study identified the molecular mechanism of miR-29b-1-5p in chicken muscle development.

Myogenetic oligodeoxynucleotide complexed with berberine promotes differentiation of chicken myoblasts.

Myoblasts are myogenic precursors that develop into myotubes during muscle formation. Improving efficiency of myoblast differentiation is important for advancing meat production by domestic animals. We recently identified novel oligodeoxynucleotides (ODNs) termed myogenetic ODNs (myoDNs) that promote the differentiation of mammalian myoblasts. An isoquinoline alkaloid, berberine, forms a complex with one of the myoDNs, iSN04, and enhances its activities. This study investigated the effects of myoDNs on chicken myoblasts to elucidate their species-specific actions. Seven myoDNs (iSN01-iSN07) were found to facilitate the differentiation of chicken myoblasts into myosin heavy chain (MHC)-positive myotubes. The iSN04-berberine complex exhibited a higher myogenetic activity than iSN04 alone, which was shown to enhance the differentiation of myoblasts into myotubes and the upregulation of myogenic gene expression (MyoD, myogenin, MHC, and myomaker). These data indicate that myoDNs promoting chicken myoblast differentiation may be used as potential feed additives in broiler diets.

Transcription of Endogenous Retrovirus Group K Members and Their Neighboring Genes in Chicken Skeletal Muscle Myoblasts

Skeletal muscle myoblasts are myogenic precursor cells that generate myofibers during muscle development and growth. We recently reported that broiler myoblasts, compared to layer myoblasts, proliferate and differentiate more actively and promptly into myocytes, which corresponds well with the muscle phenotype of broilers. Furthermore, RNA sequencing (RNA-seq) revealed that numerous genes are differentially expressed between layer and broiler myoblasts during myogenic differentiation. Based on the RNA-seq data, we herein report that chicken myoblasts transcribe endogenous retrovirus group K member (ERVK) genes. In total, 16 ERVKs were highly expressed in layer myoblasts and two (termed BrK1 and BrK2) were significantly induced in broiler myoblasts. These transcribed ERVKs had a total of 182 neighboring genes within ±100 kb on the chromosomes, of which 40% were concentrated within ±10 kb of the ERVKs. We further investigated whether the transcription of ERVKs affects the expression of their neighboring genes. BrK1 had two neighboring genes; LOC107052719 was overlapping with BrK1 and downregulated in the broiler myoblasts, and FAM19A2 was upregulated in the broiler myoblasts as well as BrK1. BrK2 had 14 neighboring genes, and only one gene, LOC772243, was differentially expressed between layer and broiler myoblasts. LOC772243 was overlapping with BrK2 and suppressed in the broiler myoblasts. These data indicate that the transcription of ERVKs may impact the expression of their neighboring genes in chicken myoblasts.

Identification of Igf2bp1 gene family and effect on chicken myoblast proliferation

ABSTRACT The function of Igf2bp1 regulated muscle development in Lueyang black-bone chickens is to be investigated. The insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) belongs to the family of single-stranded RNA-binding proteins (IGF2BP1-3). IGF2BP1 is abundantly expressed in a broad range of foetal tissues, but only in a limited number of normal adult tissues, and is involved in biological regulation processes, including cell proliferation, differentiation, metabolism and migration, especially in tumour cells. Recent study has reported Igf2bp1 was a key gene that regulated the body size of Pekin ducks. We found that the expression level of Igf2bp1 gradually decreased with age in the leg muscle of Lueyang black-bone chicken, but its function in chicken muscle development is unknown. Firstly, the evolutionary relationship of Igf2bp1 gene family was analysed in chicken. Additionally, Igf2bp1-overexpressed myoblasts of Lueyang black-bone chicken were constructed. The results showed that Igf2bp1 plays a negative role in cell proliferation by the CCK8 and 5-ethynyl-2′-deoxyuridine assay. Moreover, Igf2bp1 inhibited myogenesis by detecting the myoblast proliferation markers of pax7, the expression of myogenic differentiation markers, MyoD, MyoG and Myf5 in myoblasts. Thus, Igf2bp1 can inhibit chicken myoblast proliferation and differentiation by regulating key gene expression of myogenesis, which is not conducive to the growth and development of skeletal muscle in Lueyang black chickens.

MicroRNA-214 Inhibits Chicken Myoblasts Proliferation, Promotes Their Differentiation, and Targets the TRMT61A Gene.

The proliferation and differentiation of myoblasts is an important process of skeletal muscle development. In this process, microRNAs (miRNAs) play an important role in the proliferation and differentiation of chicken primary myoblasts (CPMs). Our previous study found that miR-214 and the tRNA methyltransferase 61A (TRMT61A) gene were differentially expressed in different stages of proliferation and differentiation. Therefore, this study aimed to explore the effect of miR-214 on the proliferation and differentiation of CPMs and the functional relationship between miR-214 and TRMT61A. In this study, we detected the effect of miR-214 on the proliferation of CPMs by qPCR, flow cytometry, CCK-8, and EdU after the overexpression and interference of miR-214. qPCR, Western blotting, and indirect immunofluorescence were used to detect the effect of miR-214 on the differentiation of the CPMs. The expression patterns of miR-214 and TRMT61A were observed at different time points of differentiation induced by the CPMs. The results show that miR-214 inhibited the proliferation of the CPMs and promoted the differentiation of the CPMs. The Dual-Luciferase Reporter assay and the expression pattern of miR-214 and TRMT61A suggested that they had a negative regulatory target relationship. This study revealed the function and regulatory mechanism of miR-214 in the proliferation and differentiation of CPMs.

Effects of Ten-Eleven Translocation-2 (Tet2) on myogenic differentiation of chicken myoblasts.

Skeletal muscle development is an orchestrated progress that is primarily regulated by temporospatial expression of myogenic regulatory factors (MRFs). Recent studies demonstrated that DNA demethylation also exerted a critical role in myogenesis. However, the function of Tet2 in the regulation of chicken myogenesis still remains unknown. In the present study, the role of Tet2 in regulating myogenic differentiation was determined by using a model of primary myoblasts from chickens. The expression of Tet2 was significantly elevated during myoblast differentiation. Meanwhile, the level of 5hmC in genomic DNA was increased, but H3K9me2 and H3K27me3 were markedly reduced following differentiation. Knockdown of Tet2 significantly inhibited the formation of multinucleated myotubes, which was accompanied by a reduction of relevant pivotal MRFs. Moreover, the level of 5hmC decreased sharply in Tet2 knockdown myoblasts. Attenuated differentiated myoblasts that resulted from reduced Tet2 also demonstrated an increased level of H3K9me2 and H3K27me3. Collectively, these results indicated that Tet2 played an essential role during myogenesis, which affected demethylation of genomic DNA and histone to regulate expression of MRFs and therefore, contributed to myoblast differentiation.

Fibromodulin Modulates Chicken Skeletal Muscle Development via the Transforming Growth Factor-β Signaling Pathway.

Simple SummaryFibromodulin (Fmod) plays critical roles in skeletal muscle development and maintenance, but the roles of Fmod in skeletal muscle atrophy and development in chickens are unclear. Here, we demonstrate that Fmod plays important roles in the differentiation and atrophy of chicken skeletal muscle by regulating the transforming growth factor-β signaling pathway. These results suggest that Fmod plays important roles in skeletal muscle growth and development in chickens.Fibromodulin (Fmod), which is an extracellular matrix protein, belongs to the extracellular matrix small-leucine-rich proteoglycan family. Fmod is abundantly expressed in muscles and connective tissues and is involved in biological regulation processes, including cell apoptosis, cell adhesion, and modulation of cytokine activity. Fmod is the main regulator of myostatin, which controls the development of muscle cells, but its regulatory path is unknown. Chicken models are ideal for studying embryonic skeletal muscle development; therefore, to investigate the mechanism of Fmod in muscle development, Fmod-silenced and Fmod-overexpressed chicken myoblasts were constructed. The results showed that Fmod plays a positive role in differentiation by detecting the expression of myogenic differentiation markers, immunofluorescence of MyHC protein, and myotube formation in myoblasts. Fmod regulates expression of atrophy-related genes to alleviate muscle atrophy, which was confirmed by histological analysis of breast muscles in Fmod-modulated chicks in vivo. Additionally, genes differentially expressed between Fmod knockdown and normal myoblasts were enriched in the signaling pathway of transforming growth factor β (TGF-β). Both Fmod-silenced and Fmod-overexpressed myoblasts regulated the expression of TGFBR1 and p-Smad3. Thus, Fmod can promote differentiation but not proliferation of myoblasts by regulating the TGF-β signaling pathway, which may serve a function in muscular atrophy.

MiRNA Profiling in Pectoral Muscle Throughout Pre- to Post-Natal Stages of Chicken Development.

MicroRNA (miRNA) is known to be an important regulator of muscle growth and development. The regulation of microRNA on the skeletal muscle phenotype of animals is mainly achieved by regulating the proliferation and differentiation of myoblasts. In this study, we sequenced a total of 60 samples from 15 developing stages of the pectoral muscle and five other tissues at 300 days of Tibetan chicken. We characterized the expression patterns of miRNAs across muscle developmental stages, and found that the chicken growth and development stage was divided into early-embryonic and late-embryonic as well as postnatal stages. We identified 81 and 21 DE-miRNAs by comparing the miRNA profiles of pectoral muscle of three broad periods and different tissues, respectively; and 271 miRNAs showed time-course patterns. Their potential targets were predicted and used for functional enrichment to understand their regulatory functions. Significantly, GgmiRNA-454 is a time-dependent and tissue-differential expression miRNA. In order to elucidate the role of gga-miRNA-454 in the differentiation of myoblasts, we cultured chicken myoblasts in vitro. The results show that although gga-miRNA-454-3p initiates increase and thereafter decrease during the chicken myoblasts differentiation, it had no effect on primary myoblasts proliferation. Furthermore, we confirm that gga-miRNA-454 inhibits myoblast differentiation by targeting the myotube-associated protein SBF2.

Functional analyses of miRNA-146b-5p during myogenic proliferation and differentiation in chicken myoblasts

BackgroundIn the poultry and livestock industries, precise genetic information is crucial for improving economic traits. Thus, functional genomic studies help to generate faster, healthier, and more efficient animal production. Chicken myoblast cells, which are required for muscle development and regeneration, are particularly important because chicken growth is closely related to muscle mass.ResultsIn this study, we induced expression of microRNA-146b-5p mediated by the piggyBac transposon system in primary chicken myoblast (pCM) cells. Subsequently, we analyzed and compared the proliferation and differentiation capacity and also examined the expression of related genes in regular pCM (rpCM) cells and pCM cells overexpressing miRNA-146b-5p (pCM-146b OE cells). pCM-146b OE cells showed increased proliferation and upregulated gene expression related to cell proliferation. In addition, next-generation sequencing analyses were performed to compare global gene expression patterns between rpCM cells and pCM-146b OE cells. We found that the higher proliferation in pCM-146b OE cells was the result of upregulation of gene sets related to the cell cycle. Moreover, miRNA-146b-5p overexpression had inhibitory effects on myotube differentiation in pCM cells.ConclusionsCollectively these results demonstrate that miR-146b-5p is closely related to the proliferation and differentiation of chicken myogenic cells as a modulator of post-transcription.

A novel transcript of MEF2D promotes myoblast differentiation and its variations associated with growth traits in chicken.

BackgroundDevelopment of skeletal muscle is closely related to broiler production traits. The myocyte-specific enhancer binding factor (MEF) 2D gene (MEF2D) and its variant transcripts play important parts in myogenesis.MethodsTo identify the transcript variants of chicken MEF2D gene and their function, this study cloned chicken MEF2D gene and identified its transcript variants from different tissue samples. The expression levels of different transcripts of MEF2D gene in different tissues and different periods were measured, and their effects on myoblast proliferation and differentiation were investigated. Variations in MEF2D were identified and association analysis with chicken production traits carried out.ResultsFour novel transcript variants of MEF2D were obtained, all of which contained highly conserved sequences, including MADS-Box and MEF2-Domain functional regions. Transcript MEF2D-V4 was expressed specifically in muscle, and its expression was increased during embryonic muscle development. The MEF2D-V4 could promote differentiation of chicken myoblasts and its expression was regulated by RBFOX2. The single nucleotide polymorphism g.36186C > T generated a TAG stop codon, caused MEF2D-V4 to terminate translation early, and was associated with several growth traits, especially on early body weight.ConclusionWe cloned the muscle-specific transcript of MEF2D and preliminarily revealed its role in embryonic muscle development.

Distinct cell proliferation, myogenic differentiation, and gene expression in skeletal muscle myoblasts of layer and broiler chickens

Myoblasts play a central role during skeletal muscle formation and growth. Precise understanding of myoblast properties is thus indispensable for meat production. Herein, we report the cellular characteristics and gene expression profiles of primary-cultured myoblasts of layer and broiler chickens. Broiler myoblasts actively proliferated and promptly differentiated into myotubes compared to layer myoblasts, which corresponds well with the muscle phenotype of broilers. Transcriptomes of layer and broiler myoblasts during differentiation were quantified by RNA sequencing. Ontology analyses of the differentially expressed genes (DEGs) provided a series of extracellular proteins as putative markers for characterization of chicken myogenic cells. Another ontology analyses demonstrated that broiler myogenic cells are rich in cell cycle factors and muscle components. Independent of these semantic studies, principal component analysis (PCA) statistically defined two gene sets: one governing myogenic differentiation and the other segregating layers and broilers. Thirteen candidate genes were identified with a combined study of the DEGs and PCA that potentially contribute to proliferation or differentiation of chicken myoblasts. We experimentally proved that one of the candidates, enkephalin, an opioid peptide, suppresses myoblast growth. Our results present a new perspective that the opioids present in feeds may influence muscle development of domestic animals.

FHL1 regulates myoblast differentiation and autophagy through its interaction with LC3.

Four and a half LIM domain protein 1 (FHL1) belongs to the FHL protein family and is predominantly expressed in skeletal and cardiac muscle. FHL1 acts as a scaffold during sarcomere assembly and plays a vital role in muscle growth and development. Autophagy is key to skeletal muscle development and regeneration, with its dysfunction associated with a range of muscular pathologies and disorders. In this study, we constructed FHL1-silenced or FHL1-overexpressed myoblasts to investigate its role in autophagy during the differentiation of chicken myoblasts into myotubules. Our data showed that FHL1 contributes to myoblast differentiation as measured through MyoG, MyoD, Myh3, and Mb mRNA expression, MyoG and MyHC protein expression and the morphological characteristics of myoblasts. The results showed that FHL1 silencing inhibited the expression of ATG5 and ATG7, meanwhile, immunofluorescence and immunoprecipitation showed that FHL1 and LC3 interacted to regulate the correct formation of autophagosomes. FHL1 inhibition increased cleaved caspase-3 and PARP abundance and promoted myoblast apoptosis. Furthermore, FHL1 rescued skeletal muscle atrophy through regulating the expression of Atrogin-1 and MuRF1. Taken together, these data suggested that FHL1 regulates chicken myoblast differentiation through its interaction with LC3.

The Inhibition on MDFIC and PI3K/AKT Pathway Caused by miR-146b-3p Triggers Suppression of Myoblast Proliferation and Differentiation and Promotion of Apoptosis.

Accumulating studies report that microRNAs (miRNAs) are actively involved in skeletal myogenesis. Previously, our study revealed that miR-146b-3p was related to the growth of skeletal muscle. Here, we further report that miR-146b-3p is essential for the proliferation, differentiation, and apoptosis of chicken myoblast. Elevated expression of miR-146b-3p can dramatically suppress proliferation and differentiation, and facilitate apoptosis of chicken myoblast. Besides, we identified two target genes of miR-146b-3p, AKT1 and MDFIC, and found that miR-146b-3p can inhibit the PI3K/AKT pathway. Our study also showed that both AKT1 and MDFIC can promote the proliferation and differentiation while inhibit the apoptosis of myoblast in chicken. Overall, our results demonstrate that miR-146b-3p, directly suppressing PI3K/AKT pathway and MDFIC, acts in the proliferation, differentiation, and apoptosis of myoblast in chicken.

Functional efficacy analysis of Angelica gigas Nakai on chicken myoblast cells through cell-based in vitro assay.

The value-added products in livestock industry is one of the key issues in order to maximize the revenue and to create a new business model. Numerous studies have suggested application of herbal plants as feed additives to increase health, productivity, and/or high-quality product in livestock. In this study, the first experiment was designed to develop in vitro evaluation system by using primary chicken myoblast (pCM) cells isolated from pectoralis major of 10-day-old male embryos. Subsequently, to evaluate effects of Korean Danggui Angelica gigas Nakai (AGN), we optimized the concentration of AGN root extract for treatment of primary pCM cells. After the treatment of AGN root extract, we compared proliferation and differentiation capacity, and also examined the gene expression. In the second experiment, the next generation sequencing analysis was performed to compare the different patterns of the global gene expression in pCM cells treated with AGN extract. Three up-regulated (pancreas beta cells, fatty acid metabolism and glycolysis) and one down-regulated (adipogenesis) gene sets were characterized suggesting that the AGN extract affected the metabolic pathways for the utilization of fat and glucose in chicken muscle cells. Furthermore, we validated the expression patterns of the up-regulated genes (GCLC, PTPN6, ISL1, SLC25A13, TGFBI, and YWHAH) in the AGN-treated pCM cells by quantitative RT-PCR. These results demonstrated that the treatment of AGN extract decreased proliferation and differentiation of pCM cells, and affected the metabolic pathways of glucose and fatty acids. Moreover, AGN extract derived from byproducts such as stem and leaf also showed the reduced proliferation patterns on AGN-treated pCM cells. Taken together, pCM cell-based in vitro assay system could be primarily and efficiently applied for evaluating the biofunctional efficacy of various feed additive candidates.