Differentiation Of Bovine Myoblasts Research Articles

Production of green-natural and “authentic” cultured meat based on proanthocyanidins-dialdehyde chitosan-collagen ternary hybrid edible scaffolds

Cultured meat has the potential to fulfill the meat demand for the growing human population, but cultured meat development will be required to simplify the production process and produce naturally cultured meat, such as no longer stripping off scaffolders and adding artificial dyes. In this study, proanthocyanidins (PC) and dialdehyde chitosan (DAC) were employed as dual crosslinkers with collagen to prepare a hybrid 3D edible scaffold for the production of high-quality cell-cultured meat. The results revealed that the scaffold was biocompatible and could offer robust mechanical support and adhesion sites for bovine myoblasts, enabling long-term cell culture. Meanwhile, the Col-PC-DAC scaffold promoted the myogenic differentiation of bovine myoblasts and extracellular matrix protein secretion, further affecting the texture of cultured meat. After cooking the cultured meat and beef, it was shown that the cultured meat had some similarities to beef in color and flavor. Importantly, our findings demonstrate that cultured meat can acquire a color remarkably similar to that of conventional beef without the need for artificial dyeing. This breakthrough not only simplifies the production process but also ensures a more natural and appealing appearance of cultured meat. In conclusion, the proanthocyanidins-dialdehyde chitosan-collagen hybrid 3D edible scaffolds provide a new option for producing cultured meat that satisfies consumer expectations.

Effects of proanthocyanidins and dialdehyde chitosan on the proliferation and differentiation of bovine myoblast for cultured meat production

Cultured meat technology intends to manufacture meat by cultivating muscle stem cells in vitro, which is an emerging methodology in meat production. However, the insufficient stemness of bovine myoblasts cultivated in vitro declined the ability of cell expansion and myogenic differentiation, which limited the production of cultured meat. Therefore, in this study, we introduced proanthocyanidins (PC, natural polyphenolic compounds) and dialdehyde chitosan (DAC, natural polysaccharides) to explore the effects of proliferation and differentiation of bovine myoblasts in vitro. The experiment results revealed that PC and DAC promoted cell proliferation by improving the transition from G1 to the S phase as well as cell division in G2. Meanwhile, the myogenic differentiation of cells was further boosted by the combined PC and DAC up-regulation of MYH3 expression. Moreover, the study revealed the synergistic effect of PC and DAC on enhancing the structural stability of collagen, and bovine myoblasts demonstrated excellent growth and dispersion ability on collagen scaffolds. It is concluded that both PC and DAC promote the proliferation and differentiation of bovine myoblasts, contributing to the development of cultured meat production systems.

Interaction of C/EBPβ with SMAD2 and SMAD4 genes induces the formation of lipid droplets in bovine myoblasts.

As a key component of Transforming growth factor-β (TGF-β) pathway, Smad2 has many crucial roles in a variety of cellular processes, but it cannot bind DNA without complex formation with Smad4. In the present study, the molecular mechanism in the progress of myogenesis underlying transcriptional regulation of SMAD2 and SMAD4 had been clarified. The result showed the inhibition between SMAD2 and SMAD4, which promotes and inhibits bovine myoblast differentiation, respectively. Further, the characterization of promoter region of SMAD2 and SMAD4 was analyzed, and identified C/EBPβ directly bound to the core region of both SMAD2 and SMAD4 genes promoter and stimulated the transcriptional activity. However, C/EBPβ has lower expression in myoblasts which plays vital function in the transcriptional networks controlling adipogenesis, while the overexpression of C/EBPβ gene in myoblasts significantly increased SMAD2 and SMAD4 gene expression, induced the formation of lipid droplet in bovine myoblasts, and promoted the expression of adipogenesis-specific genes. Collectively, our results showed that C/EBPβ may play an important role in the trans-differentiation and dynamic equilibrium of myoblasts into adipocyte cells via promoting an increase in SMAD2 and SMAD4 gene levels. These results will provide an important basis for further understanding of the TGFβ pathway and C/EBPβ gene during myogenic differentiation.

Bovine HOXA11 Gene Identified from RNA-Seq: mRNA Profile Analysis and Genetic Variation Detection Using ME Method and Their Associations with Carcass Traits.

The Homeobox A11 (HOXA11) gene regulates limb skeletal development and muscle growth, thus, it was selected as a candidate gene for bovine carcass traits. In this study, we analyzed the mRNA expression level of HOXA11 in various tissues and cells, and determined the genetic variations in the HOXA11 gene, which might be used as molecular markers for cattle breeding. The mRNA expression profiles of HOXA11 in bovine different tissues showed that HOXA11 was highly expressed in both fat and muscle. The gene expression trend of HOXA11 in myoblasts and adipocytes indicated that HOXA11 might be involved in the differentiation of bovine myoblasts and adipocytes. The data in the Ensembl database showed that there are two putative insertion/deletion (InDel) polymorphisms in the bovine HOXA11 gene. The insertion site (rs515880802) was located in the upstream region (NC_037331.1: g. 68853364-68853365) and named as P1-Ins-4-bp, and the deletion site (rs517582703) was located in the intronic region (NC_037331.1: g. 68859510-68859517) and named as P2-Del-8-bp. These polymorphisms within the HOXA11 gene were identified and genotyped by PCR amplification, agarose gel electrophoresis and DNA sequencing in the 640 Shandong Black Cattle Genetic Resource (SDBCGR) population. Moreover, the mutation frequency was very low after detection, so the mathematical expectation (ME) method was used for detection. Statistical analysis demonstrated that P1-Ins-4-bp was significantly correlated with the beef shoulder (p = 0.012) and tongue root (p = 0.004). Meanwhile, P2-Del-8-bp displayed a significant correlation with the back tendon (p = 0.008), money tendon (p = 2.84 × 10-4), thick flank (p = 0.034), beef shin (p = 9.09 × 10-7), triangle thick flank (p = 0.04), triangle flank (p = 1.00 × 10-6), rump (p = 0.018) and small tenderloin (p = 0.043) in the female SDBCGR population. In summary, these outcomes may provide a new perspective for accelerating the molecular breeding of cattle through marker-assisted selection (MAS) strategies.

Analysis of Promoter Methylation of the Bovine FOXO1 Gene and Its Effect on Proliferation and Differentiation of Myoblasts.

This study aimed to explore the regulatory role of FOXO1 promoter methylation on its transcriptional level and unravel the effect of FOXO1 on the proliferation and differentiation of bovine myoblasts. Bisulfite sequencing polymerase chain reaction (BSP) and real-time quantitative PCR were performed to determine the methylation status and transcript levels of the FOXO1 promoter region at different growth stages. BSP results showed that the methylation level in the calf bovine (CB) group was significantly higher than that in the adult bovine (AB) group (p < 0.05). On the other hand, qRT-PCR results indicated that the mRNA expression level in the AB group was significantly higher than that in the CB group (p < 0.05), suggesting a significant decrease in gene expression at high levels of DNA methylation. CCK-8 and flow cytometry were applied to determine the effect of silencing the FOXO1 gene on the proliferation of bovine myoblasts. Furthermore, qRT-PCR and Western blot were conducted to analyze the expression of genes associated with the proliferation and differentiation of bovine myoblasts. Results from CCK-8 revealed that the short hairpin FOXO1 (shFOXO1) group significantly promoted the proliferation of myoblasts compared to the short-hairpin negative control (shNC) group (p < 0.05). Flow cytometry results showed a significant decrease in the number of the G1 phase cells (p < 0.05) and a significant increase in the number of the S phase cells (p < 0.05) in the shFOXO1 group compared to the shNC group. In addition, the expression of key genes for myoblast proliferation (CDK2, PCNA, and CCND1) and differentiation (MYOG, MYOD, and MYHC) was significantly increased at both mRNA and protein levels (p < 0.05). In summary, this study has demonstrated that FOXO1 transcription is regulated by methylation in the promoter region and that silencing FOXO1 promotes the proliferation and differentiation of bovine myoblasts. Overall, our findings lay the foundation for further studies on the regulatory role of epigenetics in the development of bovine myoblasts.

Effect of bovine myosin heavy chain 3 on proliferation and differentiation of myoblast

The myosin heavy chain 3 (MYH3) gene is an essential gene that affects muscle development. This study aimed to discuss the expression characteristics of the MYH3 gene and its effect on the proliferation and differentiation of bovine myoblasts. Quantitative real time-PCR results display that the expression level of MYH3 was higher in muscle tissue, and the expression increased in the early stage of myoblast differentiation. Interfering with the MYH3 gene in myoblasts resulted in fewer EDU-positive cells and decreased expression of proliferation marker genes. Interference with MYH3 can also affect the differentiation process of myoblasts. Regarding phenotype, myotube differentiation in the interference group was slowed or even stopped. Interference with the expression of MYH3 could significantly reduce the expression of myogenic differentiation marker genes. The above results show that MYH3 is mainly expressed in muscle tissue and is highly expressed in the early stage of differentiation of bovine myoblasts, and interfering with the MYH3 can promote the proliferation and inhibit the differentiation of bovine myoblasts. This study provides a theoretical basis for revealing the regulatory process of bovine myoblast proliferation and differentiation and bovine molecular breeding.

New Insight into Muscle-Type Cofilin (CFL2) as an Essential Mediator in Promoting Myogenic Differentiation in Cattle.

Meat quality and meat composition are not separated from the influences of animal genetic improvement systems; the growth and development of skeletal muscle are the primary factors in agricultural meat production and meat quality. Though the muscle-type cofilin (CFL2) gene has a crucial influence on skeletal muscle fibers and other related functions, the epigenetic modification mechanism of the CFL2 gene regulating meat quality remains elusive. After exploring the spatiotemporal expression data of CFL2 gene in a group of samples from fetal bovine, calf, and adult cattle, we found that the level of CFL2 gene in muscle tissues increased obviously with cattle age, whereas DNA methylation levels of CFL2 gene in muscle tissues decreased significantly along with cattle age by BSP and COBRA, although DNA methylation levels and mRNA expression levels basically showed an opposite trend. In cell experiments, we found that bta-miR-183 could suppress primary bovine myoblast differentiation by negatively regulated CFL2. In addition, we packaged recombinant adenovirus vectors for CFL2 gene knockout and overexpression and found that the CFL2 gene could promote the differentiation of primary bovine myoblasts by regulating marker genes MYOD, MYOG and MYH3. Therefore, CFL2 is an essential mediator for promoting myogenic differentiation by regulating myogenic marker genes in cattle myoblasts.

MiR-183/96/182 Cluster Regulates the Development of Bovine Myoblasts through Targeting FoxO1.

Simple SummaryIn this work, we identified that the miR-183/96/182 cluster was highly expressed in bovine embryonic muscle; meanwhile, it widely existed in other organizations. Functional assays indicated that the miR-183/96/182 cluster targets the FoxO1 gene to regulate the proliferation and differentiation of bovine myoblasts.Muscle development is an important factor affecting meat yield and quality and is coordinated by a variety of the myogenic genes and signaling pathways. Recent studies reported that miRNA, a class of highly conserved small noncoding RNA, is actively involved in regulating muscle development, but many miRNAs still need to be further explored. Here, we identified that the miR-183/96/182 cluster exhibited higher expression in bovine embryonic muscle; meanwhile, it widely existed in other organizations. Functionally, the results of the RT-qPCR, EdU, CCK8 and immunofluorescence assays demonstrated that the miR-183/96/182 cluster promoted proliferation and differentiation of bovine myoblast. Next, we found that the miR-183/96/182 cluster targeted FoxO1 and restrained its expression. Meanwhile, the expression of FoxO1 had a negative correlation with the expression of the miR-183/96/182 cluster during myoblast differentiation. In a word, our findings indicated that the miR-183/96/182 cluster serves as a positive regulator in the proliferation and differentiation of bovine myoblasts through suppressing the expression of FoxO1.

Interaction of MyoD and MyoG with Myoz2 gene in bovine myoblast differentiation

This study aims to explore the functional role of Myoz2 in myoblast differentiation, and elucidate the potential factors interact with Myoz2 in promoter transcriptional regulation. The temporal-spatial expression results showed that the bovine Myoz2 gene was highest expressed in longissimus dorsi, and in individual growth stages and myoblast differentiation stages. Knockdown of Myoz2 inhibited the differentiation of myoblast, and negative effect of MyoD, MyoG, MyH and MEF2A expression on mRNA levels. Subsequently, the promoter region of bovine Myoz2 gene with 1.7 Kb sequence was extracted, and then it was set as eight series of deleted fragments, which were ligated into pGL3-basic to detect core promoter regions of Myoz2 gene in myoblasts and myotubes. Transcription factors MyoD and MyoG were identified as important cis-acting elements in the core promoter region (−159/+1). Also, it was highly conserved in different species based on dual-luciferase analysis and multiple sequence alignment analysis, respectively. Furthermore, a chromatin immunoprecipitation (ChIP) analysis combined with site-directed mutation and siRNA interference and overexpression confirmed that the combination of MyoD and MyoG occurred in region −159/+1, and played an important role in the regulation of bovine Myoz2 gene. These findings explored the regulatory network mechanism of Myoz2 gene during the development of bovine skeletal muscle.

CircNDST1 Regulates Bovine Myoblasts Proliferation and Differentiation via the miR-411a/Smad4 Axis.

Circular RNA (circRNA) is endogenous noncoding RNA found throughout the eukaryotic genome. It regulates several biological activities at the transcription or post-transcription level. However, the underlying function of circRNA in bovine skeletal muscle development remains unknown. Here, we identified a novel circRNA, circNDST1, and investigated its function and mechanism on the proliferation and differentiation of bovine myoblasts. At the molecular and cellular levels, circNDST1 could promote bovine myoblasts proliferation and inhibit differentiation. Mechanistically, circNDST1 is expressed in the cytoplasmic of myoblast and was enriched by protein Ago2. circNDST1 acts as a competing endogenous RNA that sponges miR-411a and alleviates the inhibitory effect on its target gene, Smad4. miR-411a and Smad4 were also involved in regulating bovine myoblast proliferation and differentiation. These findings suggest that circNDST1 functions as a competing endogenous RNA and regulates bovine myoblast proliferation and differentiation through the miR-411a/Smad4 axis.

CircMEF2D Negatively Regulated by HNRNPA1 Inhibits Proliferation and Differentiation of Myoblasts via miR-486-PI3K/AKT Axis.

Circular RNA (circRNA) is a form of endogenous RNA that can regulate gene expression and participate in the regulation of myogenesis. However, the molecular mechanisms and potential roles of circRNAs in bovine muscle development remain largely unknown. Nevertheless, the RNA splicing factors regulating the biogenesis of bovine circRNA have not yet been characterized. In this study, we identified a novel circRNA, circMEF2D, formed by back-splicing of constitutive exons (exons 5-7) of the bovine MEF2D gene. Functional assays showed that circMEF2D inhibited the proliferation and differentiation of bovine myoblasts. Importantly, we showed that circMEF2D regulated the PI3K-AKT signaling pathway through direct and competitive binding to miR-486. Furthermore, to explore the formation mechanism of circMEF2D, we explored the MEF2D gene alternative splicing progress. Four alternative linear variants of MEF2D were found. Due to its role in alternative splicing, the RNA-binding protein HNRNPA1 was selected for further study and the modulation of HNRNPA1 levels showed that it negatively regulated both back-splicing and linear splicing of MEF2D gene. Overall, in addition to the characterization of bovine circRNAs, these findings revealed the crucial role of HNRNPA1 in MEF2D gene alternative splicing and demonstrated a regulatory circMEF2D-miR-486-PI3K-AKT axis.

Effect of Actin Alpha Cardiac Muscle 1 on the Proliferation and Differentiation of Bovine Myoblasts and Preadipocytes.

Simple SummaryMarbling is an important factor affecting the quality of beef. The co-culture (myoblast-preadipocytes) system was successfully established in our lab in the early stage to simulate the internal environment of marbling. Within this environment, ACTC1 gene was a differentially expressed gene screened from the co-culture system. The gene was not expressed in monocultured adipocytes but was expressed in co-cultured adipocytes. Therefore, we hypothesize that the ACTC1 gene plays a role in the development of bovine myoblasts and preadipocytes. In this study, we explored the effect of ACTC1 gene on the proliferation and differentiation of bovine myoblasts and preadipocytes, aiming to discover the potential biological function of ACTC1 gene in muscle development and fat deposition. The results showed that ACTC1 could regulate the development of bovine myoblasts and preadipocytes, and ACTC1 could be used as an important target for improving beef quality in the future.Actin Alpha Cardiac Muscle 1 (ACTC1) gene is a differentially expressed gene screened through the co-culture system of myoblasts-preadipocytes. In order to study the role of this gene in the process of proliferation and differentiation of bovine myoblasts and preadipocytes, the methods of the knockdown, overexpression, and ectopic expression of ACTC1 were used in this study. After ACTC1 knockdown in bovine myoblasts and inducing differentiation, the sizes and numbers of myotube formation were significantly reduced compared to the control group, and myogenic marker genes—MYOD1, MYOG, MYH3, MRF4, MYF5, CKM and MEF2A—were significantly decreased (p < 0.05, p < 0.01) at both the mRNA and protein levels of myoblasts at different differentiation stages (D0, D2, D4, D6 and D8). Conversely, ACTC1 overexpression induced the inverse result. After ectopic expression of ACTC1 in bovine preadipocytes and induced differentiation, the number and size of lipid droplets were significantly higher than those of the control group, and the expression of adipogenic marker genes—FABP4, SCD1, PPARγ and FASN—were significantly increased (p < 0.05, p < 0.01) at the mRNA and protein levels of preadipocytes at different differentiation stages. Flow cytometry results showed that both the knockdown and overexpression of ACTC1 inhibited the normal cell cycle of myoblasts; however, ectopic expression of ACTC1 in adipocytes induced no significant cell cycle changes. This study is the first to explore the role of ACTC1 in bovine myogenesis and lipogenesis and demonstrates that ACTC1 promotes the differentiation of bovine myoblasts and preadipocytes, affecting the proliferation of myoblasts.

Transcriptome-wide N 6-Methyladenosine Methylome Profiling Reveals m6A Regulation of Skeletal Myoblast Differentiation in Cattle (Bos taurus).

N 6 -methyladenosine (m6A) is the most prevalent methylation modification of eukaryotic mRNA, and it plays an important role in regulating gene expression. Previous studies have found that m6A methylation plays a role in mammalian skeletal muscle development. However, the effect of m6A on bovine skeletal myogenesis are still unclear. Here, we selected proliferating myoblasts (GM) and differentiated myotubes (on the 4th day of differentiation, DM) for m6A-seq and RNA-seq to explore the m6A methylation modification pattern during bovine skeletal myogenesis. m6A-seq analysis revealed that m6A methylation was an abundant modification of the mRNA in bovine myoblasts and myotubes. We scanned 5,691–8,094 m6A-modified transcripts, including 1,437 differentially methylated genes (DMGs). GO and KEGG analyses revealed that DMGs were primarily involved in transcriptional regulation and RNA metabolism, as well as insulin resistance and metabolic pathways related to muscle development. The combined analysis further identified 268 genes that had significant changes at both m6A and mRNA levels, suggesting that m6A modification may regulate myoblast differentiation by mediating the expression of these genes. Furthermore, we experimentally confirmed four genes related to myogenesis, including MYOZ2, TWIST1, KLF5 and MYOD1, with differential changes in both m6A and mRNA levels during bovine myoblast differentiation, indicating that they can be potential candidate targets for m6A regulation of skeletal myogenesis. Our results may provide new insight into molecular genetics and breeding of beef cattle, and provide a reference for investigating the mechanism of m6A regulating skeletal muscle development.

MicroRNA bta-miR-365-3p inhibits proliferation but promotes differentiation of primary bovine myoblasts by targeting the activin A receptor type I

BackgroundMicroRNAs act as post-transcriptional regulators that repress translation or degrade mRNA transcripts. Each microRNA has many mRNA targets and each mRNA may be targeted by several microRNAs. Skeletal muscles express a plethora of microRNA genes that regulate muscle development and function by controlling the expression of protein-coding target genes. To expand our understanding of the role of microRNA, specifically bta-miR-365-3p, in muscle biology, we investigated its functions in regulating primary bovine myoblast proliferation and differentiation.ResultsFirstly, we found that bta-miR-365-3p was predominantly expressed in skeletal muscle and heart tissue in Chinese Qinchuan beef cattle. Quantitative PCR and western blotting results showed that overexpression of bta-miR-365-3p significantly reduced the expression levels of cyclin D1 (CCND1), cyclin dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) but stimulated the expression levels of muscle differentiation markers, i.e., MYOD1, MYOG at both mRNA and protein level. Moreover, downregulation of bta-miR-365-3p increased the expression of CCND1, CDK2 and PCNA but decreased the expression of MYOD1 and MYOG at both mRNA and protein levels. Furthermore, flow cytometry, EdU proliferation assays and immunostaining results showed that increased levels of bta-miR-365-3p suppressed cell proliferation but promoted myotube formation, whereas decreased levels of bta-miR-365-3p resulted in the opposite consequences. Finally, we identified that activin A receptor type I (ACVR1) could be a direct target of bta-miR-365-3p. It was demonstrated that bta-miR-365-3p can bind to the 3’UTR of ACVR1 gene to regulate its expression based on dual luciferase gene reporter assays. Consistently, knock-down of ACVR1 was associated with decreased expressions of CDK2, CCND1 and PCNA but increased expression of MYOG and MYOD1 both at mRNA and protein level.ConclusionCollectively, these data suggested that bta-miR-365-3p represses proliferation but promotes differentiation of bovine myoblasts through several biological mechanisms involving downregulation of ACVR1.

Effect of Neudesin Neurotrophic Factor on Differentiation of Bovine Preadipocytes and Myoblasts in a Co-Culture System.

Simple SummaryMarbling beef refers to the red and white beef formed by depositing fat between muscle fibers, and “marbling” is an important factor affecting beef quality. Therefore, we established a co-culture system of adipocytes and myocytes to mimic the microenvironment of marbling beef in vivo. However, Neudesin neurotrophic factor (NENF) was not detected in the co-culture system, but was detected in both adipocytes and myoblasts cultured separately. Further studies revealed that NENF knockdown inhibits adipogenesis and promotes myogenesis in separately cultured preadipocytes and myoblasts. However, because the monoculture system does not include the interaction of bovine adipocytes and myoblasts in the formation of marbling beef, in this study we investigated the effect of recombinant protein NENF on the differentiation of adipocytes and myoblasts in the co-culture system. The addition of NENF inhibited the formation of lipid droplets in co-cultured adipocytes but had no significant effect on myotube formation. These results were different from, and even conflicted with, those in the monocultures, which suggested that regulation of NENF expression in the same cell type changed along with the cell microenvironment and the molecular mechanism of marbling beef formation cannot be fully revealed through studies on the monoculture system.In this study, we successfully established a co-culture system of bovine preadipocytes and myoblasts to explore the effect of exogenous addition of Neudesin neurotrophic factor (NENF) recombinant protein on the differentiation of adipocytes and myoblasts in co-culture. The optimal concentration of NENF recombinant protein was 100 pg/mL. NENF promoted the differentiation of bovine preadipocytes and inhibited the differentiation of bovine myoblasts when the cells were cultured separately. After adding NENF recombinant protein to the co-culture system, the accumulation of lipid droplets in bovine preadipocytes decreased, but the differentiation of bovine myoblasts did not change significantly. The results of real-time quantitative PCR (RT-qPCR) and Western blot showed that the expression levels of adipogenesis-related factors such as PPARγ, FABP4 and FASN were significantly down-regulated at the mRNA and protein levels in adipocytes, while myogenic marker genes such as MYOD1, MYOG and MYHC had no significant changes at the mRNA or protein levels in myoblasts. This differs from, and potentially conflicts with, the monoculture system, where NENF expression in each cell type changed with the cell microenvironment. Consequently, the molecular mechanism of marbling beef formation cannot be fully revealed using monocultures of adipocytes or myocytes.

LncRNA IGF2 AS Regulates Bovine Myogenesis through Different Pathways

The role of long non-coding RNA (lncRNA) in the regulation of bovine skeletal muscle development remains poorly understood. The present study investigated the function and regulatory mechanism of a novel lncRNA, insulin-like growth factor 2 antisense transcript (IGF2 AS), in bovine myoblast proliferation and differentiation. Gain or loss of IGF2 AS was performed using an expression plasmid or small interfering RNA (siRNA), respectively. Bovine myoblasts were used to investigate the biological function and mechanisms of IGF2 AS in vitro. Results were conjointly analyzed by celluar and molecular biology experiments as well as bioinformatics. Functionally, IGF2 AS could promote proliferation and differentiation of bovine myoblasts. The preliminary mechanism suggests, on the one hand, that IGF2 AS could complement the IGF2 gene intron region and affect the stability and expression of IGF2 mRNA. On the other hand, RNA pull-down and immunoprecipitation assays demonstrated that IGF2 AS could directly bind to the interleukin enhancer binding factor 3 (ILF3) protein and maybe partly though it to regulate myogenesis. In conclusion, the novel identified lncRNA IGF2 AS promoted proliferation and differentiation of bovine myoblasts through various pathways.

MEF2A Regulates the MEG3-DIO3 miRNA Mega Cluster-Targeted PP2A Signaling in Bovine Skeletal Myoblast Differentiation.

Understanding the molecular mechanisms of skeletal myoblast differentiation is essential for studying muscle developmental biology. In our previous study, we reported that knockdown of myocyte enhancer factor 2A (MEF2A) inhibited myoblast differentiation. Here in this study, we further identified that MEF2A controlled this process through regulating the maternally expressed 3 (MEG3)—iodothyronine deiodinase 3 (DIO3) miRNA mega cluster and protein phosphatase 2A (PP2A) signaling. MEF2A was sufficient to induce MEG3 expression in bovine skeletal myoblasts. A subset of miRNAs in the MEG3-DIO3 miRNA cluster was predicted to target PP2A subunit genes. Consistent with these observations, MEF2A regulated PP2A signaling through its subunit gene protein phosphatase 2 regulatory subunit B, gamma (PPP2R2C) during bovine myoblast differentiation. MiR-758 and miR-543 in the MEG3-DIO3 miRNA cluster were down-regulated in MEF2A-depleted myocytes. Expression of miR-758 and miR-543 promoted myoblast differentiation and repressed PPP2R2C expression. Luciferase activity assay showed that PPP2R2C was post-transcriptionally targeted by miR-758 and miR-543. Taken together, these results reveal that the MEG3-DIO3 miRNAs function at downstream of MEF2A to modulate PP2A signaling in bovine myoblast differentiation.

Smad3 influences Smad2 expression via the transcription factor C/EBPα and C/EBPβ during bovine myoblast differentiation

Transforming growth factor β (TGFβ) has participated in a variety of cellular biological processes. Smad2 and Smad3 are equally important TGFβ downstream effectors in mediating TGFβ signals. However, genes involved in controlling the balance between these two signaling pathways are unknown. In this study, we showed that although Smad2 and Smad3 are structurally similar, with 89% amino acid sequence similarity in bovine, Smad3 significantly decreased Smad2 mRNA and protein expression during bovine myoblast differentiation, but not by binding on its promoter. Luciferase assays and electrophoretic mobility shift assays (EMSA) demonstrated that the transcription factors C/EBPα and C/EBPβ activate Smad2 promoter activity and expression under high serum medium (GM), whereas the opposite was observed under low serum medium (DM). Moreover, over-expression and interference assays revealed that Smad3 has a different effect on C/EBPα and C/EBPβ expression under GM versus DM conditions. After mutation of the C/EBPα and C/EBPβ binding sites, Smad3 had a reduced effect on Smad2 promoter activity. Therefore, these results demonstrated that Smad3 inhibits Smad2 expression via its transcription factors C/EBPα and C/EBPβ during bovine myoblast differentiation. This novel mechanism of the Smad2/3 genes may offer clues for further investigation of TGFβ signal function.

LncRNA-MEG3 promotes bovine myoblast differentiation by sponging miR-135.

Long noncoding RNA maternally expressed gene 3 (lncRNA-MEG3) is an important regulator in multiple biological functions. However, lncRNA-MEG3's function in cattle growth and regulatory mechanism on bovine skeletal muscle development has not yet been well studied. In this project, we first investigated lncRNA-MEG3's expression profile and detected that it was highly expressed in bovine skeletal muscle tissue and its RNA level was kept increasingly during the early phase of bovine primary myoblast differentiation. Using luciferase reporter assays, we identified the lncRNA-MEG3 core promoter containing putative transcription factor binding site for myocyte enhancer factor 2C (MEF2C). Interestingly, we found that LncRNA-MEG3 could significantly upregulate and downregulate myosin heavy chain ( MHC), myogenin ( MyoG), and MEF2C through overexpression and RNAi strategies, respectively. Using luciferase reporter assays, we also verified lncRNA-MEG3 as a miR-135 sponge. Overexpression of miR-135 markedly inhibited the wild type of lncRNA-MEG3, but not the mutant lncRNA-MEG3 reporter. The luciferase activity of miR-135 sensor could be rescued by lncRNA-MEG3 via competing for miRNA-135. In addition, the luciferase activity of MEF2C was significantly upregulated by the wild type of lncRNA-MEG3. This study, for the first time, revealed that lncRNA-MEG3 could promote bovine skeletal muscle differentiation via interacting with miRNA-135 and MEF2C. The results were valuable for further studies and applications of lncRNA related roles in beef molecular breeding.

PSXV-42 Role of cholesterol in proliferation and differentiation of bovine myoblasts.

PSXV-42 Role of cholesterol in proliferation and differentiation of bovine myoblasts.