The miR-23a~27a~24-2 cluster is an important regulator in cell metabolism. However, the cooperative and independent functions of this cluster in bovine adipocyte adipogenesis have not been elucidated. In this study, we found that expression of the miR-23a~27a~24-2 cluster was induced during adipogenesis and this cluster acted as a negative regulator of adipogenesis. miR-27a and miR-24-2 were shown to inhibit adipogenesis by directly targeting glycerol-3-phosphate acyltransferase, mitochondrial (GPAM) and diacylglycerol O-acyltransferase 2 (DGAT2), both of which promoted adipogenesis. Meanwhile, miR-23a and miR-24-2 were shown to target decorin (DCN), glucose-6-phosphate dehydrogenase (G6PD), and lipoprotein lipase (LPL), all of which repressed adipogenesis in this study. Thus, the miR-23a~27a~24-2 cluster exhibits a non-canonical regulatory role in bovine adipocyte adipogenesis. To determine how the miR-23a~27a~24-2 cluster inhibits adipogenesis while targeting anti-adipogenic genes, we identified another target gene, fibroblast growth factor 11 (FGF11), a positive regulator of adipogenesis, that was commonly targeted by the entire miR-23a~27a~24-2 cluster. Our findings suggest that the miR-23a~27a~24-2 cluster fine-tunes the regulation of adipogenesis by targeting two types of genes with pro- or anti-adipogenic effects. This balanced regulatory role of miR-23a~27a~24-2 cluster finally repressed adipogenesis.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in leptin-mediated regulation of energy metabolism. This study investigated genetic variation in STAT3 promoter regions and verified their contribution to bovine body size traits. We first estimated the degree of conservation in STAT3, followed by measurements of its mRNA expression during fetal and adult stages of Qinchuan cattle. We then sequenced the STAT3 promoter region to determine genetic variants and evaluate their association with body size traits. From fetus to adult, STAT3 expression increased significantly in muscle, fat, heart, liver, and spleen tissues (p < 0.01), but decreased in the intestine, lung, and rumen (p < 0.01). We identified and named five single nucleotide polymorphisms (SNPs): SNP1-304A>C, SNP2-285G>A, SNP3-209A>C, SNP4-203A>G, and SNP5-188T>C. These five mutations fell significantly outside the Hardy–Weinberg equilibrium (HWE) (Chi-squared test, p < 0.05) and significantly associated with body size traits (p < 0.05). Individuals with haplotype H3H3 (CC-GG-CC-GG-CC) were larger in body size than other haplotypes. Therefore, variations in the STAT3 gene promoter regions, most notably haplotype H3H3, may benefit marker-assisted breeding of Qinchuan cattle.

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.

Neudesin neurotrophic factor (NENF) is a secreted protein that is essential in multiple biological processes, including neural functions, adipogenesis, and tumorigenesis. In our previous study, NENF was significantly inhibited in the bovine adipocytes-myoblasts co-culture system. However, studies on NENF regulation of bovine muscle development and involvement in the cross-talk between adipose tissue and skeletal muscle have not been reported. Hence, the aim of this study was to clarify the functional roles of NENF in bovine preadipocytes and myoblasts. Real-time quantitative PCR (RT-qPCR) was performed to examine the spatial expression patterns of NENF in different tissues, and the results showed that NENF was highly expressed in the muscle of four-day-old and 24-month-old Qinchuan cattle. Compared with four-day-old Qinchuan cattle, the expression level of NENF was significantly up-regulated in 24-month-old bovine adipose tissue. To explore the roles of NENF in bovine myoblast and preadipocyte differentiation, small interfering RNA (siRNA) targeting the NENF gene were transfected into bovine preadipocytes and myoblasts to knock down the expression of the NENF gene. The results showed that the knockdown of NENF in differentiating adipocytes attenuated lipid accumulation, decreased the mRNA expression of adipogenic key marker genes PPARγ, CEBPα, CEBPβ, FASN, and SCD1, and decreased the protein expression of PPARγ, CEBPα, and FASN. The formation of myotubes was significantly accelerated, and the mRNA expression levels of myogenic marker genes MYOD1, MYF5, MYF6, MEF2A, MEF2C, and CKM, and the protein expression levels of MYOD1, MYF6, MEF2A, and CKM were up-regulated in myoblasts where NENF was knocked down. In short, the knockdown of NENF inhibited preadipocyte differentiation and promoted myoblast myogenesis.

Tumor protein p53 inducible nuclear protein 2 (TP53INP2) is a key positive regulator of autophagy, and it has been shown to modulate adipocyte differentiation. However, the molecular mechanism involved in autophagy regulation during adipocyte differentiation has not been clarified. Our experiments were intended to investigate whether TP53INP2 is involved in the regulation of autophagy during bovine adipocyte differentiation and how TP53INP2 affects the differentiation of bovine adipocytes. In our research, using RT-qPCR and Western blot methods, we found that the overexpression of TP53INP2 resulted in the upregulation of adipogenesis and autophagy-related genes, and autophagy flux and the degree of differentiation were detected by LipidTOX™ Deep Red Neutral Lipid staining and dansylcadaverine staining, respectively. The knockdown of TP53INP2 produced results that were the inverse of those produced by the overexpression of TP53INP2. Overall, our results suggested that TP53INP2 can activate autophagy during the early stage of differentiation in bovine adipocytes and positively regulate adipocyte differentiation by affecting autophagy. Additionally, peroxisome proliferator-activated receptor gamma (PPARγ) also contributed to the function of TP53INP2 in modulating adipocyte differentiation.

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.