Bovine Skeletal Muscle Satellite Cells Research Articles

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.

Lnc133b, a novel, long non-coding RNA, regulates bovine skeletal muscle satellite cell proliferation and differentiation by mediating miR-133b

The proliferation and differentiation of skeletal muscle satellite cells is regulated by multiple regulatory factors including non-coding RNAs. It has been reported that miR-133b regulates myogenesis. In this study, we detected a novel lncRNA, lnc133b, which is completely complemented by mature miR-133b, indicating that lnc133b may regulate the expression of miR-133b by “sponge” miR-133b. A luciferase report assay confirmed that lnc133b interacts with miR-133b in regions complemented by miR-133b. We successfully constructed lnc133b gain/loss-of-function cell models by infecting LV-1nc133b and transfecting si-lnc133b into satellite cells. Results of quantitative real-time polymerase chain reaction (qRT-PCR) and 5-ethynyl-2′-deoxyuridine (EdU) assays showed that overexpression or inhibition of lnc133b could promote the proliferation or inhibition of satellite cell differentiation. The qRT-PCR results also showed that lnc133b negatively regulates miR-133b expression and a Western blot assay showed that lnc133b positively regulates IGF1R expression, indicating that the lnc133b/miR-133b/IGF1R axis is a potential pathway for promoting satellite cell proliferation and repressing their differentiation through the ceRNA mechanism. Building on the findings of previous reports, we constructed the lnc133b/miR-133b/FGFR1 & PP2AC pathway to improve the lnc133b regulation network regulating the proliferation and differentiation of satellite cells. The current study provides a new perspective for understanding the mechanism regulating satellite cell proliferation and differentiation through the interaction of miR-133b and lnc133b.

LncRNA H19 promotes the differentiation of bovine skeletal muscle satellite cells by suppressing Sirt1/FoxO1

BackgroundH19 is a well-characterized Long noncoding RNA (lncRNA) that has been proven to promote myoblast differentiation in humans and mice. However, its mechanism of action is still not fully interpreted.MethodsUsing RT-qPCR, we examined H19 RNA levels in various tissues from 1-week, 1-month, 6-month and 36-month old male cattle (i.e., newborn, infant, young and adult). The protein and mRNA levels of MyoG, MyHC, Sirt1 and FoxO1 in the satellite and C2C12 cells with an H19 silencing or overexpression vector were respectively detected using western blot and real-time qPCR.ResultsH19 was highly expressed in skeletal muscle at all the studied ages. High expression of H19 was required for the differentiation of bovine satellite cells. Knockdown of H19 caused a remarkable increase in the myoblast-inhibitory genes Sirt1/FoxO1, suggesting that H19 suppresses Sirt1/FoxO1 expression during myogenesis. Western blotting analysis of co-transfection of Sirt1 or FoxO1 expression vectors with pcDNA-H19 indicated that Sirt1/FoxO1 overexpression neutralized the promotion of myoblast differentiation through transfection of pcDNA-H19.ConclusionH19 promoted the differentiation of bovine skeletal muscle satellite cells by suppressing Sirt1/FoxO1.

Transcription factor EGR1 promotes differentiation of bovine skeletal muscle satellite cells by regulating MyoG gene expression.

The transcription factor, early growth response 1 (EGR1), has important roles in various cell types in response to different stimuli. EGR1 is thought to be involved in differentiation of bovine skeletal muscle-derived satellite cells (MDSCs); however, the precise effects of EGR1 on differentiation of MDSCs and its mechanism of action remain unknown. In the present study, a time course of EGR1 expression and the effects of EGR1 on MDSC differentiation were determined. The results demonstrated that the expression of EGR1 mRNA and protein increased significantly in differentiating MDSCs relative to that in proliferating cells. Over-expression of the EGR1 gene in MDSCs promoted their differentiation and inhibited proliferation. Conversely, knock-down of EGR1 inhibited differentiation of MDSCs and promoted their proliferation, indicating that EGR1 promotes MDSC differentiation. Moreover, over-expression of EGR1 in MDSCs increased the expression of MyoG mRNA and protein, whereas its knock-down had the opposite effect. Furthermore, ChIP-PCR analyses demonstrated that EGR1 could bind directly to its putative binding site within the promoter region of MyoG, and determination of ERG1 subcellular localization in MDSCs demonstrated that it could relocate to the nucleus, indicating MyoG is likely an EGR1 target gene whose expression is positively regulated by this transcription factor. In conclusion, EGR1 can promote MDSC differentiation through positive regulation of MyoG gene expression.

Impact of Bovine Skeletal Muscle Satellite Cell Differentiation by Small Interfering RNA Targeting Myogenin Gene

To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, then used cell transfection, real time RCR and Western Blot to detect the influence of myogenin to cell differentiation. Results showed that the knockdown of myogenin significantly decreased its expression and other muscle-specific genes. Compared to the control, it could differentiate into few myotubes when challenged by low serum in the medium. These findings provided an important theoretical basis for further explore of the genetic mechanism in adult skeletal muscle, the remedy of muscle injuries and the cultivation of high-yield transgenic cattle.

Effect of TCEA3 on the differentiation of bovine skeletal muscle satellite cells

Bovine muscle-derived satellite cells (MDSCs) are important for animal growth. In this study, the effect of transcription elongation factor A3 (TCEA3) on bovine MDSC differentiation was investigated. Western blotting, immunofluorescence assays, and cytoplasmic and nuclear protein isolation and purification techniques were used to determine the expression pattern and protein localization of TCEA3 in bovine MDSCs during in vitro differentiation. TCEA3 expression was upregulated using the CRISPR/Cas9 technique to study its effects on MDSC differentiation in vitro. TCEA3 expression gradually increased during the in vitro differentiation of bovine MDSCs and peaked on the 5th day of differentiation. TCEA3 was mainly localized in the cytoplasm of bovine MDSCs, and its expression was not detected in the nucleus. The level of TCEA3 was relatively higher in myotubes at a higher degree of differentiation than during early differentiation. After transfection with a TCEA3-activating plasmid vector (TCEA3 overexpression) for 24 h, the myotube fusion rate, number of myotubes, and expression levels of the muscle differentiation-related loci myogenin (MYOG) and myosin heavy chain 3 (MYH3) increased significantly during the in vitro differentiation of bovine MDSCs. After transfection with a TCEA3-inhibiting plasmid vector for 24 h, the myotube fusion rate, number of myotubes, and expression levels of MYOG and MYH3 decreased significantly. Our results indicated, for the first time, that TCEA3 promotes the differentiation of bovine MDSCs and have implications for meat production and animal rearing.

MiR-143 regulates proliferation and differentiation of bovine skeletal muscle satellite cells by targeting IGFBP5.

Development of skeletal muscle is a complicated biological process regulated by various regulation factors and signal pathways. MicroRNAs (miRNAs) are novel gene regulators that control muscle cell development. microRNA-143 (miR-143) is highly expressed in skeletal muscle, and we found that miR-143 level is significantly increased during bovine skeletal muscle satellite cells (MSCs) differentiation process through microarray analysis and qRT-PCR detection. However, the function of miR-143 in bovine muscle development remained unclear. In our work, the functions of miR-143 in bovine MSCs myogenic differentiation were investigated. We discovered that IGFBP5 is directly regulated by miR-143 using a dual-luciferase reporter assay. Overexpression of miR-143 led to decreased level of IGFBP5 protein and restrained cell proliferation and differentiation, while downregulation of miR-143 resulted in increased levels of IGFBP5 protein and restrained cell proliferation but improved differentiation. IGFBP5, an important component of IGF signaling pathway, contributes greatly to bovine muscle cell development. A mechanism that miR-143 can regulate the proliferation and differentiation of bovine MSCs through changing expression of IGFBP5 was elucidated by our study.

Effect of differentiation on microRNA expression in bovine skeletal muscle satellite cells by deep sequencing.

BackgroundThe differentiation of skeletal muscle-derived satellite cells (MDSCs) is important in controlling muscle growth, improving livestock muscle quality, and healing of muscle-related disease. MicroRNAs (miRNAs) are a class of gene expression regulatory factors, which play critical roles in the regulation of muscle cell differentiation. This study aimed to compare the expression profile of miRNAs in MDSC differentiation, and to investigate the miRNAs which are involved in MDSC differentiation.MethodTotal RNA was extracted from MDSCs at three different stages of differentiation (MDSC-P, MDSC-D1 and MDSC-D3, representing 0, 1 and 3 days after differentiation, respectively), and used to construct small RNA libraries for RNA sequencing (RNA-seq).ResultsThe results showed that in total 617 miRNAs, including 53 novel miRNA candidates, were identified. There were 9 up-expressed, 165 down-expressed, and 15 up-expressed, 145 down-expressed in MDSC-D1 and MDSC-D3, respectively, compared to those in MDSC-P. Also, 17 up-expressed, 55 down-expressed miRNAs were observed in MDSC-D3 compared to those in MDSC-D1. All known miRNAs belong to 237 miRNA gene families. Furthermore, we observed some sequence variants and base edits of the miRNAs. GO and KEGG pathway analysis showed that the majority of target genes regulated by miRNAs were involved in cellular metabolism, pathways in cancer, actin cytoskeleton regulation and the MAPK signaling pathway. Regarding the 53 novel miRNAs, there were 7 up-expressed, 31 down-expressed, and 8 up-expressed, 26 down-expressed in MDSC-D1 and MDSC-D3, respectively, compared to those in MDSC-P. The expression levels of 12 selected miRNA genes detected by RT-qPCR were consistent with those generated by deep sequencing.ConclusionsThis study confirmed the authenticity of 564 known miRNAs and identified 53 novel miRNAs which were involved in MDSC differentiation. The identification of novel miRNAs has significantly expanded the repertoire of bovine miRNAs and could contribute to advances in understanding muscle development in cattle.Electronic supplementary materialThe online version of this article (doi:10.1186/s11658-016-0009-x) contains supplementary material, which is available to authorized users.

MicroRNA-128 regulates the proliferation and differentiation of bovine skeletal muscle satellite cells by repressing Sp1.

MicroRNAs (miRNAs) play essential roles in muscle cell proliferation and differentiation. The muscle-specific miRNAs miR-1 and miR-206 have been shown to regulate muscle development and promote myogenic differentiation; however, it is likely that a number of other miRNAs play important roles in regulating myogenesis as well. microRNA-128 (miR-128) has been reported to be highly expressed in brain and skeletal muscle, and we found that miR-128 is also up-regulated during bovine skeletal muscle satellite cell differentiation using microarray analysis and qRT-PCR. However, little is known about the functions of miR-128 in bovine skeletal muscle satellite cell development. In this study, we investigated the biological functions of miR-128 in bovine skeletal muscle cell development. Using a dual-luciferase reporter assay, we confirmed that miR-128 regulates the Sp1 gene. Over-expression of miR-128 reduced Sp1 protein levels and inhibited muscle satellite cell proliferation and differentiation. Inhibition of miR-128 increased Sp1 protein levels and promoted muscle satellite cell differentiation but also suppressed proliferation. Changes in miR-128 and Sp1 expression levels also affected the protein levels of MyoD and CDKN1A. Sp1, an activator of MyoD and a suppressor of CDKN1A, plays an important role in bovine muscle cell proliferation and differentiation. The results of our study reveal a mechanism by which miR-128 regulates bovine skeletal muscle satellite cell proliferation and myogenic differentiation via Sp1.

The role of microRNA-1 and microRNA-206 in the proliferation and differentiation of bovine skeletal muscle satellite cells.

MicroRNAs (miRNAs) have been found to play essential roles in muscle cell proliferation and differentiation. MicroRNA-1 (miR-1) and microRNA-206 (miR-206), which are similar and have the same seed sequence, have specific roles in modulating skeletal muscle proliferation and differentiation in vitro and in vivo. However, there is no information about their function during bovine skeletal muscle satellite cell development. In this study, the profiles of miR-1 and miR-206 and their biological functions in bovine skeletal muscle cell development was investigated. The target genes were predicted, and we used a dual-luciferase reporter assay to demonstrate that miR-1 and miR-206 directly targeted the 3' untranslated region (3'UTR) of paired-box transcription factor Pax7 and histone deacetylase 4 (HDAC4). We showed that miR-1 and miR-206 facilitate bovine skeletal muscle satellite cell myogenic differentiation by restricting the expression of their target gene and that inhibition of miR-1 and miR-206 increased the Pax7 and HDAC4 protein levels and substantially enhanced satellite cell proliferation. Therefore, our results revealed the mechanism in which miR-1 and miR-206 positively regulate bovine skeletal muscle satellite cell myogenic differentiation via Pax7 and HDAC4 downregulation.

Identification and bioinformatics analysis of miRNAs involved in bovine skeletal muscle satellite cell myogenic differentiation.

MicroRNAs (miRNAs) are short non-coding RNA molecules that perform post-transcriptional repression of target genes by binding to 3' untranslated regions, and involved in the regulation of many biological processes. Some studies indicate that miRNAs are mechanistically involved in the muscle growth and differentiation. However, little is known about miRNAs expression patterns during the process of bovine skeletal muscle satellite cell myogenic differentiated into myotubes. To investigate the mechanisms of miRNAs-mediated regulation during this process, we performed a miRNAs microarray to detect 783 bovine miRNAs in bovine skeletal muscle satellite cell myogenic differentiation, and the results were further confirmed by a quantitative real-time RT-PCR assay. We observed that the expression of 15 miRNAs was significantly different between bovine skeletal muscle satellite cells and differentiated myotubes, in which twelve were significantly up-regulated and three were down-regulated in myotubes. Furthermore, using bioinformatics methods, the targets of differentially expressed miRNAs were predicted, and were further subjected to gene ontology (GO) and KEGG analysis. A total of 3077 potential target genes were produced, and the highly enriched GOs and KEGG pathways showed that these genes together formed a regulatory network that involved in cell proliferation, cell differentiation, and multiple biological molecular signaling processes. Taken together, the results of the current study suggested the potential regulating roles of these differentially expressed miRNAs in bovine myogenic differentiation.

Effect of MSTN Propeptide and shRNA Co-expression Vector on Proliferation of Skeletal Muscle Satellite Cells

Myostatin (MSTN) is a negative regulator of skeletal muscle growth, in order to study the effect of inhibition MSTN expression on the proliferation of bovine skeletal muscle satellite cells, we constructed co-expression vector pcDNA3.1-Pro-MSTNshRNA, transfected it into muscle satellite cells by Liposome 2000, and detected cell proliferation changes by CCK-8 method and flow cytometry after 48 h. The expressions of P21 and CDK2 were detected by Western blot and real-time PCR. The results showed that the cell vitality of experimental groups significantly increased than that of the negative control, and cells in S phase also increased significantly (P<0.05). After knocked down MSTN gene, P21 expression decreased (P<0.05), but CDK2 gene expression increased (P<0.05). These results indicated that MSTN gene expression was associated with P21 and CDK2, the proliferation of skeletal muscle satellite cells could be promoted while MSTN was inhibited, which provided a theoretical basis for the study on transgenic cattle.

Effects of CMV Enhancer on Activity and Specificity of Bovine MyoG Gene Promoter

Connected a segment of CMV enhancer to the front of MyoG gene promoter and then constructed the corresponding dual luciferase expression vector pGL3-CMV-MyoGpro. We set four eukaryotic expression vectors including pGL3-CMV, pGL3-MyoGpro, pGL3-CMV-MyoGpro, and pGL3-Basic which contained CMV promoter, MyoG promoter, CMV-MyoG synthesis promoter, and a promoterless negative control, respectively. Then the four vectors and internal control Renilla luciferase report gene vector phRL-TK were transfected into bovine skeletal muscle satellite cells, mouse C2C12 cells and bovine fetal fibroblast cells to detect the promoter activity with dual luciferase report system. The results showed that CMV enhancer could significantly improve the transcription activity of bovine MyoG gene promoter in muscle satellite cells and mouse C2C12 cells, and it had certain specificity. This study provided experimental materials for increasing the high expression of exogenous gene in bovine muscle cells, and also laid the molecular theoretical basis for obtaining the high specific promoter of bovine muscle and the transgenic beef cattle.

Significant role of μ-calpain (CANP1) in proliferation/survival of bovine skeletal muscle satellite cells

Calpains are a family of Ca2+-dependent intracellular cysteine proteases, including the ubiquitously expressed μ-calpain (CANP1) and m-calpain (CANP2). The CANP1 has been found to play a central role in postmortem proteolysis and meat tenderization. However, the physiological roles of CANP1 in cattle skeletal satellite cells remain unclear. In this study, three small interference RNA sequences (siRNAs) targeting CANP1 gene were designed and ligated into pSilencer plasmid vector to construct shRNA expression constructs. Suppression of CANP1 in satellite cells was evaluated using these shRNA expressing constructs. Our results revealed that all three siRNAs could downregulate the expression of CANP1. Suppression of CANP1 significantly reduced cell viability in cell proliferation when compared with control cells. We found a crosstalk between CANP1 and caspase systems, particularly suppression of CANP1 resulted in an increase in the expressions of apoptotic caspases such as caspase-3, caspase-6, caspase-7, caspase-8, and caspase-9, as well as heat-shock protein (HSP) systems. Additionally, suppression of CANP1 led to the upregulation of other apoptosis and DNA damage-regulating genes whilst at the same time downregulating proliferation, migration, and differentiation-regulating genes. The results of our findings report for the first time that suppression of CANP1 resulted in the activation of caspase and HSP systems which might in turn regulate apoptosis through the caspase-dependent cell death pathway. This clearly demonstrates the key roles of CANP1 in regulation of cell proliferation and survival.Electronic supplementary materialThe online version of this article (doi:10.1007/s11626-013-9666-5) contains supplementary material, which is available to authorized users.