Differentiation Of Skeletal Muscle Satellite Cells Research Articles

MiR-21-5p Regulates the Proliferation and Differentiation of Skeletal Muscle Satellite Cells by Targeting KLF3 in Chicken.

The proliferation and differentiation of skeletal muscle satellite cells (SMSCs) play an important role in the development of skeletal muscle. Our previous sequencing data showed that miR-21-5p is one of the most abundant miRNAs in chicken skeletal muscle. Therefore, in this study, the spatiotemporal expression of miR-21-5p and its effects on skeletal muscle development of chickens were explored using in vitro cultured SMSCs as a model. The results in this study showed that miR-21-5p was highly expressed in the skeletal muscle of chickens. The overexpression of miR-21-5p promoted the proliferation of SMSCs as evidenced by increased cell viability, increased cell number in the proliferative phase, and increased mRNA and protein expression of proliferation markers including PCNA, CDK2, and CCND1. Moreover, it was revealed that miR-21-5p promotes the formation of myotubes by modulating the expression of myogenic markers including MyoG, MyoD, and MyHC, whereas knockdown of miR-21-5p showed the opposite result. Gene prediction and dual fluorescence analysis confirmed that KLF3 was one of the direct target genes of miR-21-5p. We confirmed that, contrary to the function of miR-21-5p, KLF3 plays a negative role in the proliferation and differentiation of SMSCs. Si-KLF3 promotes cell number and proliferation activity, as well as the cell differentiation processes. Our results demonstrated that miR-21-5p promotes the proliferation and differentiation of SMSCs by targeting KLF3. Collectively, the results obtained in this study laid a foundation for exploring the mechanism through which miR-21-5p regulates SMSCs.

PDLIM5 Affects Chicken Skeletal Muscle Satellite Cell Proliferation and Differentiation via the p38-MAPK Pathway.

Simple SummaryPDZ and LIM domain 5 (PDLIM5) can increase C2C12 cell differentiation; however, the role of PDLIM5 in chicken skeletal muscle satellite cells (SMSCs) is unclear. In this study, the effect of PDLIM5 was verified on SMSCs in vitro, and then the molecular mechanism was determined by transcriptome sequencing. We demonstrated that PDLIM5 can positively affect chicken SMSC proliferation and differentiation via the p38-MAPK (mitogen activated kinase-like protein) pathway. These results indicate that PDLIM5 may be involved in chicken skeletal muscle growth and development.Skeletal muscle satellite cell growth and development is a complicated process driven by multiple genes. The PDZ and LIM domain 5 (PDLIM5) gene has been proven to function in C2C12 myoblast differentiation and is involved in the regulation of skeletal muscle development. The role of PDLIM5 in chicken skeletal muscle satellite cells, however, is unclear. In this study, in order to determine whether the PDLIM5 gene has a function in chicken skeletal muscle satellite cells, we examined the changes in proliferation and differentiation of chicken skeletal muscle satellite cells (SMSCs) after interfering and overexpressing PDLIM5 in cells. In addition, the molecular pathways of the PDLIM5 gene regulating SMSC proliferation and differentiation were analyzed by transcriptome sequencing. Our results show that PDLIM5 can promote the proliferation and differentiation of SMSCs; furthermore, through transcriptome sequencing, it can be found that the differential genes are enriched in the MAPK signaling pathway after knocking down PDLIM5. Finally, it was verified that PDLIM5 played an active role in the proliferation and differentiation of chicken SMSCs by activating the p38-MAPK signaling pathway. These results indicate that PDLIM5 may be involved in the growth and development of chicken skeletal muscle.

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

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.

Long Noncoding RNA (lncRNA) CTTN-IT1 Elevates Skeletal Muscle Satellite Cell Proliferation and Differentiation by Acting as ceRNA for YAP1 Through Absorbing miR-29a in Hu Sheep.

Characterizing the factors that regulate the growth and development of muscle is central to animal production. Skeletal muscle satellite cells (SMSCs) provide an important material for simulating the proliferation and differentiation of muscle cells. YAP1, which can promote muscle growth, is closely related to the proliferation of SMSCs in Hu sheep (Ovis aries). In addition, some miRNAs, such as miR-541-3p, miR-142-5p, and miR-29a, can play critical roles in muscle growth by specifically binding with their target mRNAs. Meanwhile, lncRNA can competitively bind these miRNAs and reduce the regulatory effect of miRNAs on their target genes and thus play critical roles themselves in muscle growth. However, the regulatory molecular mechanism of miRNA and lncRNA on SMSC proliferation through YAP1 remains unclear. Here, we characterized the regulatory network among YAP1 and its targeted miRNAs and lncRNAs in Hu sheep SMSCs. The potential ncRNAs that regulate YAP1 (miR-29a and CTTN-IT1) were predicted through multilevel bioinformatics analysis. Dual-luciferase assays, RT-qPCR, and western blots revealed that miR-29a can significantly reduce the mRNA and protein expression level by binding to a specific 3′-UTR of YAP1 (P < 0.05), while CTTN-IT1 can restore the expression of YAP1 through competitive binding to miR-29a. Furthermore, the mRNA and protein expression levels of MyoG, MyoD, and MyHC showed that miR-29a can inhibit the expression of genes related to the differentiation of SMSCs, and CTTN-IT1 can increase the expression of these same genes. Thus, miR-29a may inhibit the differentiation of SMSCs and CTTN-IT1 can restore this inhibition. The EdU staining assay indicated that excessive miR-29a can significantly reduce the proliferation ability of SMSCs (P < 0.05), while overexpression of CTTN-IT1 can significantly increase the proliferation of SMSCs (P < 0.01). CTTN-IT1 is a novel lncRNA that is a competing endogenous RNA (ceRNA) of miR-29a and can promote SMSC proliferation and differentiation by restoring the expression of YAP1 when it is inhibited by miR-29a in Hu sheep. Overall, our findings construct a CTTN-IT1-miR-29a-YAP1 regulatory network that will help contribute new insight into improving the muscle development of Hu sheep.

Gga-miR-3525 Targets PDLIM3 through the MAPK Signaling Pathway to Regulate the Proliferation and Differentiation of Skeletal Muscle Satellite Cells.

MicroRNAs (miRNAs) are evolutionarily conserved, small noncoding RNAs that post-transcriptionally regulate expression of their target genes. Emerging evidence demonstrates that miRNAs are important regulators in the development of skeletal muscle satellite cells (SMSCs). Our previous research showed that gga-miR-3525 is differentially expressed in breast muscle of broilers (high growth rate) and layers (low growth rate). In this study, we report a new role for gga-miR-3525 as a myogenic miRNA that regulates skeletal muscle development in chickens. Exogenous increases in the expression of gga-miR-3525 significantly inhibited proliferation and differentiation of SMSCs, whereas the opposite effects were observed in gga-miR-3525 knockdown SMSCs. We confirmed that PDLIM3 (PDZ and LIM domain 3) is a target gene of gga-miR-3525 that can promote proliferation and differentiation of SMSCs. We found that PDLIM3 overexpression elevated the abundance of phosphorylated (p-)p38 protein but that the gga-miR-3525 mimic and p38-MAPK inhibitor (SB203580) weakened the activation of p-p38. Furthermore, treatment with SB203580 reduced the promoting effect of PDLIM3 on SMSC proliferation and differentiation. Overall, our results indicate that gga-miR-3525 regulates the proliferation and differentiation of SMSCs by targeting PDLIM3 via the p38/MAPK signaling pathway in chickens.

MiR-148a-3p Regulates Skeletal Muscle Satellite Cell Differentiation and Apoptosis via the PI3K/AKT Signaling Pathway by Targeting Meox2

As bioinformatic approaches have been developed, it has been demonstrated that microRNAs (miRNAs) are involved in the formation of muscles and play important roles in regulation of muscle cell proliferation and differentiation. Previously, it has been demonstrated that miR-148a-3p is one of the most abundant miRNAs in chicken skeletal muscle. Here, we build on that work and demonstrate that miR-148a-3p is important in the control of differentiation of chicken skeletal muscle satellite cells (SMSCs). Elevated expression of miR-148a-3p significantly promoted differentiation and inhibited apoptosis of SMSCs but did not affect proliferation. Furthermore, it was observed that the mesenchyme homeobox 2 (Meox2) is a target gene of miR-148a-3p and that miR-148a-3p can down-regulate expression of Meox2, which promote differentiation of SMSCs and suppress apoptosis. Furthermore, miR-148a-3p overexpression encouraged activation of the PI3K/AKT signaling pathway, which could be recovered by overexpression of Meox2. Overall, these findings suggest that microRNA-148a-3p is a potent promoter of myogenesis via direct targeting of Meox2 and increase of the PI3K/AKT signaling pathway in chicken SMSCs.

A novel lncRNA, lnc403, involved in bovine skeletal muscle myogenesis by mediating KRAS/Myf6.

Skeletal muscle, the most abundant and plasticity tissue in mammals, is essential for various functions such as movement, breathing, maintaining posture and metabolism. Myogenesis is a complex and precise process, which is regulated by the sequential expression of multiple transcription factors, and accumulating evidence have confirmed that multiple lncRNAs are involved in muscle development as the important transcriptional regulator. In this study, a novel lncRNA, named lnc403 was obtained, with a full-length 2689bp, which had poor coding ability and was mainly expressed in the nucleus of myoblasts and myotubes. The expression of lnc403 was significantly different in the proliferation and differentiation stages of muscle cells. Then we successfully constructed lnc403 loss/gain-function cell models by transfecting silnc403 and pCDNA3.1-EGFP-lnc403 into satellite cells, respectively; and found that lnc403 inhibited skeletal muscle satellite cell differentiation but had no significant effect on cell proliferation, either in the case of lnc403 knockdown or overexpression. In order to further screen the target factors regulated by lncRNA in the process of myogenic differentiation, the RNA-pull down, mass spectrometry and bioinformatics analysis were performed. The results showed that lnc403 negatively regulated the expression of the adjacent gene Myf6 and positively regulated interaction proteins KRAS expression. The above results indicate that lnc403 affects skeletal muscle cell differentiation by affecting the expression of nearby genes and interacting proteins, implying lnc403 might participate in the bovine myoblasts differentiation through multi-pathway network regulation mode. This study provides a new perspective for further understanding of the regulation mechanism of lncRNAs on bovine myogenic process.

MicroRNA Profiling Reveals an Abundant miR-200a-3p Promotes Skeletal Muscle Satellite Cell Development by Targeting TGF-β2 and Regulating the TGF‑β2/SMAD Signaling Pathway.

MicroRNAs (miRNAs) are evolutionarily conserved, small noncoding RNAs that play critical post-transcriptional regulatory roles in skeletal muscle development. Chicken is an optimal model to study skeletal muscle formation because its developmental anatomy is similar to that of mammals. In this study, we identified potential miRNAs in the breast muscle of broilers and layers at embryonic day 10 (E10), E13, E16, and E19. We detected 1836 miRNAs, 233 of which were differentially expressed between broilers and layers. In particular, miRNA-200a-3p was significantly more highly expressed in broilers than layers at three time points. In vitro experiments showed that miR-200a-3p accelerated differentiation and proliferation of chicken skeletal muscle satellite cells (SMSCs) and inhibited SMSCs apoptosis. The transforming growth factor 2 (TGF-β2) was identified as a target gene of miR-200a-3p, and which turned out to inhibit differentiation and proliferation, and promote apoptosis of SMSCs. Exogenous TGF-β2 increased the abundances of phosphorylated SMAD2 and SMAD3 proteins, and a miR-200a-3p mimic weakened this effect. The TGF-β2 inhibitor treatment reduced the promotional and inhibitory effects of miR-200a-3p on SMSC differentiation and apoptosis, respectively. Our results indicate that miRNAs are abundantly expressed during embryonic skeletal muscle development, and that miR-200a-3p promotes SMSC development by targeting TGF-β2 and regulating the TGF-β2/SMAD signaling pathway.

MiR-99a-5p Regulates the Proliferation and Differentiation of Skeletal Muscle Satellite Cells by Targeting MTMR3 in Chicken.

Noncoding RNAs, especially microRNAs (miRNAs), have been reported to play important roles during skeletal muscle development and regeneration. Our previous sequencing data revealed that miR-99a-5p is one of the most abundant miRNAs in chicken breast muscle. The purpose of this study was to reveal the regulatory mechanism of miR-99a-5p in the proliferation and differentiation of chicken skeletal muscle satellite cells (SMSCs). Through the investigation of cell proliferation activity, cell cycle progression, and 5-ethynyl-29-deoxyuridine (EdU) assay, we found that miR-99a-5p can significantly promote the proliferation of SMSCs. Moreover, we found that miR-99a-5p can inhibit myotube formation by decreasing the expression of muscle cell differentiation marker genes. After miR-99a-5p target gene scanning, we confirmed that miR-99a-5p directly targets the 3′ untranslated region (UTR) of myotubularin-related protein 3 (MTMR3) and regulates its expression level during chicken SMSC proliferation and differentiation. We also explored the role of MTMR3 in muscle development and found that its knockdown significantly facilitates the proliferation but represses the differentiation of SMSCs, which is opposite to the effects of miR-99a-5p. Overall, we demonstrated that miR-99a-5p regulates the proliferation and differentiation of SMSCs by targeting MTMR3.

Molecular characterization, tissue distribution, and functional analysis of the STAC3 gene in chicken.

The Src homology 3 and cysteine-rich domain 3 gene (STAC3) encodes a protein containing both a cysteine-rich domain and two Src (sarcoma) homology 3 domains (SH3). STAC3 is specifically expressed in skeletal muscle and plays an important role in skeletal muscle development, but the explicit sequence and function of chicken SATC3 remain unknown. In the current study, we found the full-length chicken STAC3 cDNA to be 1383bp long, with a 1092bp open reading frame that harbors one cysteine-rich C1 domain and two SH3 domains. Tissue distribution analysis reveals chicken STAC3 mRNA only in skeletal muscle among 12 chicken tissues examined by reverse transcription PCR. Both cholecystokinin octapeptide analysis and a 5-ethynyl-2'-deoxyuridine assay suggest that neither STAC3 overexpression nor knockdown has any effect on the proliferation of chicken skeletal muscle satellite cells. However, STAC3 knockdown significantly increases the mRNA expression of MyoG, MyoD, Mb, and MyHC, and the protein abundance of MyHC and MyoG, whereas the opposite result is found in STAC3 overexpressed cells. We conclude that the STAC3 gene is expressed specifically in skeletal muscle and is a negative regulator of skeletal muscle satellite cell differentiation in chicken.

MiR-9-5p Inhibits Skeletal Muscle Satellite Cell Proliferation and Differentiation by Targeting IGF2BP3 through the IGF2-PI3K/Akt Signaling Pathway.

MicroRNAs are evolutionarily conserved, small non-coding RNAs that play critical post-transcriptional regulatory roles in skeletal muscle development. We previously found that miR-9-5p is abundantly expressed in chicken skeletal muscle. Here, we demonstrate a new role for miR-9-5p as a myogenic microRNA that regulates skeletal muscle development. The overexpression of miR-9-5p significantly inhibited the proliferation and differentiation of skeletal muscle satellite cells (SMSCs), whereas miR-9-5p inhibition had the opposite effect. We show that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is a target gene of miR-9-5p, using dual-luciferase assays, RT-qPCR, and Western Blotting, and that it promotes proliferation and differentiation of SMSCs. In addition, we found that IGF2BP3 regulates IGF-2 expression, using overexpression and knockdown studies. We show that Akt is activated by IGF2BP3 and is essential for IGF2BP3-induced cell development. Together, our results indicate that miR-9-5p could regulate the proliferation and differentiation of myoblasts by targeting IGF2BP3 through IGF-2 and that this activity results in the activation of the PI3K/Akt signaling pathway in skeletal muscle cells.

The effect of lncRNA TCONS_00815878 on differentiation of porcine skeletal muscle satellite cells

Porcine skeletal muscle development is a complex biological process, and differentiation of skeletal muscle satellite cells is an important part of skeletal muscle development. In recent years, it has been found that lncRNA plays an important role in the differentiation of skeletal muscle satellite cells. Here we investigate the effect of lncRNA TCONS_00815878 on the differentiation of porcine skeletal muscle satellite cells. We first used qRT-PCR to detect the expression levels of TCONS_00815878 in six tissues (heart, spleen, lung, kidney, back muscles and leg muscles) of Yorkshire piglets within seven days of birth. At the same time, the expression levels of TCONS_00815878 at five different time points from the embryonic stage to the postnatal stage (35 d, 45 d, 55 d of embryos, and 7 d, 200 d of postpartum leg muscles) were examined. The expression of the differentiation marker genes MyoD, MyoG and MyHC was examined by knocking down TCONS_00815878 in porcine skeletal muscle satellite cells using antisense oligonucleotides (ASO). The target gene of TCONS_00815878 was predicted by bioinformatics analysis, and the function and pathway of its target gene were predicted online using DAVID software. The results showed that TCONS_00815878 had the highest expression level in pig myocardium and leg muscles. Within seven days after birth, TCONS_00815878 increased in the muscle tissue of pigs, and reached the peak of expression level on the 7th day. During the process of proliferation and differentiation of porcine skeletal muscle satellite cells, the expression level of TCONS_00815878 increased during the differentiation stage and peaked at 30 h of differentiation. After knocking down TCONS_00815878, the expression levels of MyoD, MyoG and MyHC were decreased, but the expression level of MyoD was significantly decreased (P<0.05). In addition, functional predictions revealed that the target gene of TCONS_00815878 is enriched in multiple biological processes, such as glycolysis and pyruvate metabolism, related to skeletal muscle satellite cell differentiation. This study speculates that lncRNA TCONS_00815878 may promote the differentiation of porcine skeletal muscle satellite cells.

Long Noncoding Ribonucleic Acid MSTRG.59589 Promotes Porcine Skeletal Muscle Satellite Cells Differentiation by Enhancing the Function of PALLD.

Skeletal muscle satellite cells are a class of undifferentiated mononuclear myogenic stem cells distributed between the myofibroblast and membrane basement. Since their development determines the development of skeletal muscles, knowledge of their proliferation, differentiation, and fate is vital for understanding skeletal muscle development. Increasing evidence have shown that long noncoding RNA (lncRNA) plays an important role in regulating the development process of satellite cells. Based on the results of our previous studies, we screened lncRNA MSTRG.59589, which is highly expressed in skeletal muscle tissue. In the present study, knockdown of MSTRG.59589 significantly inhibited satellite cell differentiation at various time points, whereas overexpression of MSTRG.59589 demonstrated opposite effects. An MSTRG.59589 knockdown cell model was constructed for transcriptome sequencing, and RNA sequencing analysis screened out a large number of differentially expressed genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of these differentially expressed genes revealed that they are mainly enriched in actin cytoskeleton, muscle contraction, and other pathways related to muscle development. Mechanistic analyses showed that MSTRG.59589 could promote the differentiation process of skeletal muscle satellite cells by positively regulating the expression level of the target gene PALLD. This experiment lays a theoretical foundation for deeper studies on the mechanism of MSTRG.59589 in the differentiation of porcine skeletal muscle satellite cells.

A Novel Long Noncoding RNA, lncR-125b, Promotes the Differentiation of Goat Skeletal Muscle Satellite Cells by Sponging miR-125b.

Long noncoding RNAs (lncRNAs) have emerged as essential regulators of skeletal myogenesis, but few myogenesis-associated lncRNAs have been identified and our understanding of their regulatory mechanisms remains limited, particularly in goat. Here, we identified a novel lncRNA, TCONS_00006810 (named lncR-125b), from our previous lncRNA sequencing data on fetal (45, 60, and 105 days of gestation, three biological replicates for each point) and postnatal (3 days after birth, n = 3) goat skeletal muscle, and found that it is highly expressed in skeletal muscle and gradually upregulated during skeletal muscle satellite cell (SMSC) differentiation in goat. Notably, overexpression of lncR-125b accelerated the expression of myogenic differentiation 1 (MyoD 1) and myogenin (MyoG), and the formation of myotubes, and knockdown of lncR-125b showed opposite effects in SMSCs. Results of dual-luciferase assay and quantitative real-time polymerase chain reaction revealed that lncR-125b acts as a molecular sponge for miR-125b and that insulin-like growth factor 2 (IGF2), a critical regulator of skeletal myogenesis, is a direct target gene of miR-125b. Further analyses showed that lncR-125b negatively regulates miR-125b expression and positively regulates IGF2 expression in SMSCs. Mechanistically, lncR-125b promotes SMSC differentiation by functioning as a competing endogenous RNA (ceRNA) for miR-125b to control IGF2 expression. These findings identify lncR-125b as a novel noncoding regulator of muscle cell differentiation and skeletal muscle development in goat.

Glycogen synthase kinase 3β (GSK3β) regulates the expression of MyHC2a in goat skeletal muscle satellite cells (SMSCs).

Glycogen synthase kinase beta (GSK3β) plays an important role in skeletal muscle growth, regeneration, and repair. However, the mechanism of GSK3β regulating MyHC2a expression is currently not clear. In this study, GSK3β inhibition promoted skeletal muscle satellite cells (SMSCs) differentiationand increased expression of MyoD, MyoG, MyHC1, and MyHC2a genes. Then we cloned approximately 1.1kb of goat MyHC2a gene promoter. The deletion fragment (-514/+55) of MyHC2a promoter exhibited the highest level of promoter activity, and a NFATc2 element in this region was responsible for MyHC2a promoter activity. Treatment of SB216713 significantly decreased the transcriptional activity of the fragment (-514/+55). Furthermore, GSK3β inhibition had no effect on the luciferase activity of MyHC2a promoter after mutating the NFATc2-binding site. These results demonstrated that GSK3β inhibition promoted SMSCs differentiation and regulated the MyHC2a gene expression through NFATc2 in goat-differentiated SMSCs.

PDH-mediated metabolic flow is critical for skeletal muscle stem cell differentiation and myotube formation during regeneration in mice.

Skeletal muscle satellite cells (SMSCs), the major stem cells responsible for the regeneration of skeletal muscle, are normally cell cycle arrested but differentiate to generate myocytes upon muscle damage, forming new myofibers along with self-renewing stem cells in preparation for subsequent injury. In this study, we investigated which factors stimulate the proliferation and differentiation of SMSCs and found that pyruvate, the end product of glycolysis, stimulates their differentiation. Pyruvate antagonizes the effects of hypoxia on preferential self-renewal of SMSCs through dephosphorylation or activation of pyruvate dehydrogenase (PDH), which mediates opening of the gateway from glycolysis to the tricarboxylic acid (TCA) cycle by producing acetyl coenzyme A from pyruvate. PDH kinase 1, highly expressed under hypoxia, is down-regulated under normoxic conditions, leading to an increase in dephosphorylated PDH. Conditional deletion of PDH in SMSCs affects cell divisions generating myocytes and subsequent myotube formation, inefficient skeletal muscle regeneration upon injury, and aggravated pathogenesis of a dystrophin-deficient mouse model of Duchenne muscular dystrophy. Thus, the flow from glycolysis to the TCA cycle mediated by PDH plays a pivotal role in the differentiation of SMSCs, which is critical for the progression of skeletal muscle regeneration.-Hori, S., Hiramuki, Y., Nishimura, D., Sato, F., Sehara-Fujisawa, A. PDH-mediated metabolic flow is critical for skeletal muscle stem cell differentiation and myotube formation during regeneration in mice.

Circular RNA profiling identified an abundant circular RNA circTMTC1 that inhibits chicken skeletal muscle satellite cell differentiation by sponging miR-128-3p.

Scope: Myogenesis involves a series of complex cellular and developmental processes regulated by many genes, transcription factors and non-coding RNAs. Recent studies have demonstrated the involvement of circular RNAs (circRNAs) in myogenesis. While previous studies have established a role for some circRNAs, the precise functions and mechanisms of circRNAs in skeletal muscle development are still not completely understood in chicken.Methods: To identify potential circRNAs during chicken embryonic skeletal muscle development, rRNA- libraries sequencing was performed in breast muscles from 12 broilers and 12 layers at four different embryonic points, embryonic day 10 (E10), E13, E16 and E19. Through circRNA differential expression analysis and target miRNA prediction, the circTMTC1 was predicted to participate in the embryonic muscle formation by sponging miRNA, which were verified in vitro experiments.Results: We identified 228 differentially expressed circRNAs between broilers and layers (fold change >2; p-value < 0.05), and 43 circRNAs were differentially expressed at multiple embryonic days. circTMTC1, a novel circRNA transcribed from the TMTC1 gene, was expressed significantly higher in layers than in broilers at E10, E13 and E16. Furthermore, circTMTC1 knockdown accelerated proliferation and differentiation in chicken skeletal muscle satellite cells (SMSCs), besides, circTMTC1-overexpressing cells showed opposite effects. circTMTC1 functioned as a miR-128-3p sponge at the differentiation stage of SMSCs, and circTMTC1 inhibited the expression of miR-128-3p. Furthermore, miR-128-3p promoted differentiation of chicken SMSCs, and circTMTC1 inhibited the promotion effect of miR-128-3p on chicken SMSC differentiation.Conclusion: Our study revealed that circRNAs are differentially expressed during chicken embryonic development between the two chicken models, and circTMTC1 inhibits chicken SMSC differentiation by sponging miR-128-3p.

Fatty acids promote bovine skeletal muscle satellite cell differentiation by regulating ELOVL3 expression.

Fatty acids (FAs) play essential roles in regulating differentiation and proliferation by affecting gene expression in various cell types. However, their potential functions in bovine cells remain unclear. Herein, we examine the differentiation and proliferation of bovine skeletal muscle-derived satellite cells (MDSCs) after incubation with three types of representative FAs (palmitic acid, oleic acid and docosahexaenoic acid) by western blotting, immunofluorescence assays, flow cytometry analysis and EdU incorporation assays. The myotube fusion rate, myotube length and expression levels of muscle differentiation-related gene myogenin (MYOG) and myosin heavy chain 3 (MYH3) increased significantly, although the FAs did not affect proliferation. Additionally, FA-induced bovine MDSC differentiation increased ELOVL3 expression and relocation of ELOVL3 to cytoplasmic lipid droplets in the differentiation of bovine MDSCs. Moreover, the effect of FAs on bovine MDSC differentiation was inhibited upon ELOVL3 downregulation. Collectively, these data indicate that FAs promote bovine MDSC differentiation by regulating ELOVL3 expression.

Molecular and functional characterization of the adiponectin (AdipoQ) gene in goat skeletal muscle satellite cells.

ObjectiveIt is commonly accepted that adiponectin binds to its two receptors to regulate fatty acid metabolism in adipocytes. To better understand their functions in the regulation of intramuscular adipogenesis in goats, we cloned the three genes (adiponectin [AdipoQ], adiponectin receptor 1 [AdipoR1], and AdipoR2) encoding these proteins and detected their mRNA distribution in different tissues. We also determined the role of AdipoQ in the adipogenic differentiation of goat skeletal muscle satellite cells (SMSCs).MethodsSMSCs were isolated using 1 mg/mL Pronase E from the longissimus dorsi muscles of 3-day-old female Nanjiang brown goats. Adipogenic differentiation was induced in satellite cells by transferring the cells to Dulbecco’s modified Eagle’s medium supplemented with an isobutylmethylxanthine, dexamethasone and insulin cocktail. The pEGFP-N1-AD plasmid was transfected into SMSCs using Lipofectamine 2000. Expression of adiponectin in tissues and SMSCs was detected by quantitative polymerase chain reaction and immunocytochemical staining.ResultsThe three genes were predominantly expressed in adipose and skeletal muscle tissues. According to fluorescence and immunocytochemical analyses, adiponectin protein expression was only observed in the cytoplasm, suggesting that adiponectin is localized to the cytoplasm of goat SMSCs. In SMSCs overexpressing the AdipoQ gene, adiponectin promoted SMSC differentiation into adipocytes and significantly (p<0.05) up-regulated expression of AdipoR2, acetyl-CoA carboxylase, fatty-acid synthase, and sterol regulatory element-binding protein-1, though expression of CCAAT/enhancer-binding protein-α, peroxisome proliferator-activated receptor γ, and AdipoR1 did not change significantly.ConclusionAdiponectin induced SMSC differentiation into adipocytes, indicating that adiponectin may promote intramuscular adipogenesis in goat SMSC.

Recent advances on the role of long non-coding RNA H19 in regulating mammalian muscle growth and development.

As one of the first identified long non-coding RNAs (lncRNAs), H19 plays a wide range of roles in vivo, including not only as a tumor suppressor and oncogene involved in disease process, but also as a regulator of growth and development of multiple tissues in mammalian embryos. The function of H19 in muscles (both skeletal and cardiac muscle) draws widespread attention due to the following two reasons. On one hand, H19 promotes myogenic differentiation and myogenesis of skeletal muscle satellite cells (SMSCs) via regulating Igf2 in cis. On the other hand, H19 also modulates the target genes in trans, including sponging let-7, miR-106 or miR-29 to mediate myocyte glucose uptake, cardiomyocyte proliferation and tendon repair, as well as promote embryonic development and muscle regeneration through binding to MBD1 as a chromatin modifier. In this review, we summarize the role of H19 in mammalian muscles, which will provide a reference for further research to unveil the molecular mechanism of muscle growth and development.