Bovine Muscle-derived Satellite Cells Research Articles

WISP1 promotes bovine MDSC differentiation via recruitment of ANXA1 for the regulation of the TGF-β signalling pathway.

Skeletal muscle is one of the most important tissues of the human body necessary for sporting activities. The differentiation of muscle-derived satellite cells (MDSCs) plays an important role in the development and regeneration of skeletal muscles. Similarly, the Wnt/β-catenin signalling pathway plays an important role in the process of muscle differentiation. Wnt1-inducible signalling pathway protein-1 (WISP1), a downstream protein of the Wnt/β-catenin signalling pathway and a member of the CCN family that also plays an important role in the differentiation process, and its expression increase during the differentiation of bovine MDSCs. However, its role in MDSC differentiation is poorly understood. Therefore, we investigated the mechanisms regulating this process via Western blot and immunofluorescence staining. Immunoprecipitation and mass spectrometry detected annexin A1 (ANXA1), a protein that interacts with WISP1. To determine whether WISP1 influences TGF-β signalling and differentiation independently of ANXA1, the latter was knocked down, while WISP1 was activated. WISP1 expression increased significantly during bovine MDSC differentiation. However, WISP1 did not affect the TGF-β signalling pathway protein marker when ANXA1 was inhibited. Taken together, WISP1 regulates the TGF-β signalling pathway through ANXA1 recruitment, thereby promoting bovine MDSC differentiation, suggesting the Wnt/β-catenin signalling pathway as another target to promote cell differentiation.

Podocan Promotes Differentiation of Bovine Skeletal Muscle Satellite Cells by Regulating the Wnt4-β-Catenin Signaling Pathway.

BackgroundSmall leucine-rich repeat proteins (SLRPs) are highly effective and selective modulators of cell proliferation and differentiation. Podocan is a newly discovered member of the SLRP family. Its potential roles in the differentiation of bovine muscle-derived satellite cells (MDSCs) and its underlying functional mechanism remain unclear. Our study aimed to characterize the function of the podocan gene in the differentiation of bovine MDSCs and to clarify the molecular mechanism by which podocan functions in order to contribute to a better understanding of the molecular mechanism by which extracellular matrix promotes bovine MDSC differentiation and provide a theoretical basis for the improvement of beef quality.MethodsBovine MDSCs were transfected with vectors to overexpress or inhibit podocan, and podocan protein was added to differentiation culture medium. qRT-PCR, western blotting, and immunofluorescence were performed to investigate the effects of podocan on MDSC differentiation. Confocal microscopy and western blotting were used to assess the nuclear translocation and expression of β-catenin. An inhibitor and activator of β-catenin were used to assess the effects of the Wnt/β-catenin signaling pathway on MDSC differentiation. We inhibited β-catenin while overexpressing podocan in MDSCs. Then, we performed mass spectrometry to identify which proteins interact with podocan to regulate the Wnt/β-catenin signaling pathway. Finally, we confirmed the relationship between podocan and Wnt4 by co-immunoprecipitation and western blotting.ResultsPodocan protein expression increased significantly during bovine MDSC differentiation. Differentiation of bovine MDSC was promoted and suppressed by podocan overexpression or inhibition, respectively. Podocan was also shown to modulate the Wnt/β-catenin signaling pathway. Treatment of bovine MDSCs with β-catenin inhibitor and activator showed that the Wnt/β-catenin pathway is involved in bovine MDSC differentiation. Furthermore, the effect of podocan on bovine MDSC differentiation was suppressed when this pathway was inhibited. We also found that podocan interacts with Wnt4. When Wnt4 was inhibited, podocan-induced promotion of bovine MDSC differentiation was attenuated through Wnt/β-catenin signaling.ConclusionPodocan regulates Wnt/β-catenin through Wnt4 to promote bovine MDSC differentiation.

WDR13 promotes the differentiation of bovine skeletal muscle-derived satellite cells by affecting PI3K/AKT signaling.

Muscle satellite cells are usually at rest, and when externally stimulated or regulated, they can be further differentiated by cell fusion to form new myotubes and muscle fibers. WD repeat domain 13 (WDR13) is highly conserved in vertebrates. Studies have shown that mice lacking the Wdr13 gene develop mild obesity, hyperinsulinemia, and increased islet β cell proliferation. However, the role of WDR13 in bovine cells is unclear. Here, we investigated the effect of WDR13 on bovine skeletal muscle-derived satellite cells (MDSCs). We found that WDR13 was upregulated in bovine MDSCs using western blotting and immunofluorescence experiments. Moreover, activation and inhibition of WDR13 expression increased and decreased cell differentiation, respectively, suggesting that WDR13 promotes bovine MDSC differentiation. To further understand the mechanism of action of WDR13, we examined changes in the PI3K/AKT signaling pathway following WDR13 activation or inhibition. In addition, cells were treated with a phosphoinositide kinase 3 (PI3K) inhibitor, LY294004, to observe cell differentiation. The results showed that activation of WDR13 inhibited the PI3K/AKT signaling pathway and enhanced cell differentiation. These data suggest that WDR13 can promote the differentiation of bovine MDSCs by affecting the PI3K/AKT signaling pathway.

MiR-139 promotes differentiation of bovine skeletal muscle-derived satellite cells by regulating DHFR gene expression.

MicroRNAs play an important regulatory role in the proliferation and differentiation of skeletal muscle-derived satellite cells (MDSCs). In particular, miR-139 can inhibit tumor cell proliferation and invasion, and its expression is down-regulated during C2C12 myoblast differentiation. The aim of this study was thus to examine the effect and potential mechanism of miR-139 in bovine MDSCs. The expression of miR-139 was found to be significantly increased during bovine MDSC differentiation by stem-loop reverse transcription-polymerase chain reaction amplification. Statistical analysis of the myotube fusion rate was done through immunofluorescence detection of desmin, and western blotting was used to measure the change in protein expression of the muscle differentiation marker genes MYOG and MYH3. The results showed that the miR-139 mimic could enhance the differentiation of bovine MDSCs, whereas the inhibitor had the opposite effect. By using the dual-luciferase reporter system, miR-139 was found to target the 3'-untranslated region of the dihydrofolate reductase (DHFR) gene and regulate its expression. In addition, the expression of miR-139 was found to be regulated by its host gene phosphodiesterase 2A (PDE2A) via inhibition of the latter by CRISPR interference (CRISPRi). Overall, our findings indicate that miR-139 plays an important role in regulating the differentiation of bovine MDSCs.

Effects of COL8A1 on the proliferation of muscle-derived satellite cells.

Collagen type VIII alpha 1 chain (COL8A1) is a component of the extracellular matrix. Our previous studies suggested that COL8A1 is associated with the proliferation of muscle-derived satellite cells (MDSCs). Additionally, it has been demonstrated that COL8A1 promotes the proliferation of smooth muscle cells and liver cancer cells. Therefore, we predicted that COL8A1 is associated with the proliferation of bovine MDSCs, which have potential applications in research. In this study, we constructed vectors to activate and repress COL8A1 in bovine MDSCs using the CRISPR/Cas9 technique and determined the effects of COL8A1 modulation by EdU labeling, Western blotting, and dual-luciferase reporter assays. The results showed that activation of COL8A1 increased the number of EdU-positive cells and expression of the proliferation markers cyclin B1 (CCNB1) and P-AKT. The expression of P-Akt was unchanged after addition of LY294002 (a protein kinase inhibitor capable of blocking the signal transduction pathway of the phosphoinositide 3-kinase). In contrast, repression of COL8A1 reduced the number of EdU-positive cells and expression of CCNB1 and P-AKT. We also observed upregulation and downregulation of COL8A1 following the overexpression and repression of EGR1, respectively. The dual-luciferase reporter assay revealed that EGR1 regulates the promoter activity of COL8A1. To our knowledge, this is the first study demonstrating that EGR1 positively regulates the expression of COL8A1, which in turn promotes the proliferation of bovine MDSCs via the PI3 K/AKT signaling pathway.

Effect of ECM2 expression on bovine skeletal muscle-derived satellite cell differentiation.

Extracellular matrix components have important regulatory functions during cell proliferation and differentiation. In recent study, extracellular matrix were shown to have a strong effect on skeletal muscle differentiation. Here, we aimed to elucidate the effects of extracellular matrix protein 2 (ECM2), an extracellular matrix component, on the differentiation of bovine skeletal muscle-derived satellite cells (MDSCs). Western blot and immunofluorescence analyses were used to elucidate the ECM2 expression pattern in bovine MDSCs during differentiation in vitro. CRISPR/Cas9 technology was used to activate or inhibit ECM2 expression to study its effects on the in vitro differentiation of bovine MDSCs. ECM2 expression was shown to increase gradually during bovine MDSC differentiation, and the levels of this protein were higher in more highly differentiated myotubes. ECM2 activation promoted MDSC differentiation, whereas its suppression inhibited the differentiation of these cells. Here, for the first time, we demonstrated the importance of ECM2 expression during bovine MDSC differentiation; these results could lead to treatments that help to increase beef cattle muscularity.

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.

Effect of ELOVL3 expression on bovine skeletal muscle-derived satellite cell differentiation

ELOVL3 is involved in elongating saturated and monounsaturated fatty acids, and is a critical enzyme for lipid accumulation in brown adipocytes during the early phase of tissue recruitment. In addition, ELOVL3 is related to increased fatty acid oxidation in brown adipocytes. However, the potential functions of ELOVL3 in bovine cells remain unclear. Herein, we aimed to elucidate the effect of the ELOVL3 on the differentiation of bovine skeletal muscle-derived satellite cells (MDSCs). Western blot and immunofluorescence analyses were used for elucidating ELOVL3 expression pattern in bovine MDSCs during differentiation in vitro. We activated or inhibited ELOVL3 to study the effect of alterations in its expression on in vitro differentiation of bovine MDSCs. ELOVL3 expression increased gradually during bovine MDSC differentiation, and its levels were higher in the more highly differentiated myotubes. Activation of ELOVL3 promoted MDSC differentiation, while inhibition of ELOVL3 hindered differentiation of these cells. Here, for the first time, we demonstrate the importance of ELOVL3 during bovine MDSC differentiation, which may assist in increasing beef cattle muscularity.

MiR-2425-5p targets RAD9A and MYOG to regulate the proliferation and differentiation of bovine skeletal muscle-derived satellite cells

Our group previously identified miR-2425-5p, a unique bovine miRNA; however, its biological function and regulation in muscle-derived satellite cells (MDSCs) remain unclear. Herein, stem-loop RT-PCR results showed that miR-2425-5p increased during MDSCs proliferation, but decreased during differentiation. Cell proliferation was examined using EdU assays, cyclin B1 (CCNB1) and proliferating cell nuclear antigen (PCNA) western blot (WB) and flow cytometry analysis. These results showed that miR-2425-5p mimics (miR-2425-M) enhanced MDSCs proliferation, whereas, miR-2425-5p inhibitor (miR-2425-I) had opposite effect. Conversely, cell differentiation studies by desmin (DES) immunofluorescence, myotubes formation, and myosin heavy chain 3 (MYH3) WB analyses revealed that miR-2425-M and miR-2425-I blocked and promoted MDSCs differentiation, respectively. Moreover, luciferase reporter, RT-PCR, and WB assays showed that miR-2425-5p directly targeted the 3′-UTR of RAD9 homolog A (RAD9A) and myogenin (MYOG) to regulate their expression. Rescue experiment showed RAD9A inhibited the proliferation of MDSCs through miR-2425-5p. In addition, we found that miR-2425-5p expression was regulated by its host gene NCK associated protein 5-like (NCKAP5L) rather than being transcribed independently as a separate small RNA. Collectively, these data indicate that miR-2425-5p is a novel regulator of bovine MDSCs proliferation and differentiation and provides further insight into the biological functions of miRNA in this species.

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.

Transcriptional profiling of bovine muscle-derived satellite cells during differentiation in vitro by high throughput RNA sequencing.

In this study, we utilized high throughput RNA sequencing to obtain a comprehensive gene expression profile of muscle-derived satellite cells (MDSCs) upon induction of differentiation. MDSCs were cultured in vitro and RNA was extracted for sequencing prior to differentiation (MDSC-P), and again during the early and late differentiation (MDSC-D1, and MDSC-D3, respectively) stages. Sequence tags were assembled and analyzed by digital gene expression profile to screen for differentially expressed genes, Gene Ontology annotation, and pathway enrichment analysis. Quantitative real-time PCR was used to confirm the results of RNA sequencing. Our results indicate that certain of genes were changed during skeletal muscle cell development, cell cycle progression, and cell metabolism during differentiation of bovine MDSCs. Furthermore, we identified certain genes that could be used as novel candidates for future research of muscle development. Additionally, the sequencing results indicated that lipid metabolism might be the predominant cellular process that occurs during MDSC differentiation.