Proteomics insights into the effects of MSTN on muscle glucose and lipid metabolism in genetically edited cattle.

The molecular mechanism underlying myostatin (MSTN)-regulated metabolic cross-talk remains poorly understood. In this study, we performed comparative proteomic and phosphoproteomic analyses of gluteus muscle tissues from MSTN-/- transgenic cattle using a shotgun-based tandem mass tag (TMT) 6-plex labeling method to explore the signaling pathway of MSTN in metabolic cross-talk and cellular metabolism during muscle development. A total of 72 differentially expressed proteins (DEPs) and 36 differentially expressed phosphoproteins (DEPPs) were identified in MSTN-/- cattle compared to wild-type cattle. Bioinformatics analyses showed that MSTN knockout increased the activity of many key enzymes involved in fatty acid ß-oxidation and glycolysis processes in cattle. Furthermore, comprehensive pathway analyses and hypothesis-driven AMP-activated protein kinase (AMPK) activity assays suggested that MSTN knockout triggers the activation of AMPK signaling pathways to regulate glucose and lipid metabolism by increasing the AMP/ATP ratio. Our results shed new light on the potential regulatory mechanism of MSTN associated with metabolic cross-talk in muscle development, which can be used in animal breeding to improve meat production in livestock animals, and can also provide valuable insight into treatments for obesity and diabetes mellitus in humans.

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.

Insights into the function of n-3 PUFAs in fat-1 transgenic cattle.

The n-3 PUFAs have many beneficial effects on human health, including roles in immunity, neurodevelopment, and preventing cardiovascular disease. In this study, we established reliable model fat-1 transgenic cattle using transgenic technology and performed a systematic investigation to examine the function of n-3 PUFAs. Our results showed that expression of the fat-1 gene improved several biochemical parameters related to liver function and to plasma glucose and plasma lipid metabolism. Results of global gene and plasma protein expression analysis showed that 310 genes and 13 plasma proteins differed significantly in the blood of fat-1 transgenic cattle compared with WT cattle, reflecting their regulatory roles in the immune and cardiovascular systems. Finally, changes in the gut microflora were also noted in the fat-1 transgenic cattle, suggesting novel roles for n-3 PUFAs in the metabolism of glucose and lipids, as well as anti-stress properties. To the best of our knowledge, this is the first report using multiple parallel analyses to investigate the role of n-3 PUFAs using models such as fat-1 transgenic cattle. This study provides novel insights into the regulatory mechanism of fat-1 in the immune and cardiovascular systems, as well as its anti-stress role.

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.

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.

Enhanced expression of MYF5 and MYOD1 in fibroblast cells via the forced expression of bos taurus MYF5.

The formation of vertebrate skeletal muscles widely thought to be under the control of hierarchy of regulatory genes. MYF5 is one of the myogenic determination gene expressed in the developing mouse dermomyotome which control skeletal muscle differentiation. In the current work, we had obtained the cDNA sequence including the full coding region of the bos taurus myogenic factor MYF5 by reverse transcription polymerase chain reaction. Furthermore, we examined whether fibroblast cell derived from mouse and bos taurus can be transduced using plasmid vectors carrying bos taurus MYF5. Bos taurus MYF5 activates MYF5 and MYOD1 expression after 1 day culture. The concerted upregulation of the myogenic regulatory factors enhanced myosin (skeletal fast) expression. These observation show that MYF5 is essential for myogenic differentiation and provides candidates for regulation bos taurus skeletal muscle development.