Growing evidence has revealed that microRNA are central elements in milk fat synthesis in mammary epithelial cells. A negative regulator of adipocyte fat synthesis, miR-27a has been reported to be involved in the regulation of milk fat synthesis in goat mammary epithelial cells; however, the regulatory role of miR-27a in bovine milk fat synthesis remains unclear. In the present study, primary bovine mammary epithelial cells (BMEC) were harvested from mid-lactation cows and cultured in Dulbecco's modified Eagle's medium/F-12 medium with 10% fetal bovine serum, 5 μg/mL of insulin, 1 μg/mL of hydrocortisone, 2 μg/mL of prolactin, 1 μg/mL of progesterone, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. We found that the overexpression of miR-27a significantly suppressed lipid droplet formation and decreased the cellular triacylglycerol (TAG) levels, whereas inhibition of miR-27a resulted in a greater lipid droplet formation and TAG accumulation in BMEC. Meanwhile, overexpression of miR-27a inhibited mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG), CCAAT/enhancer-binding protein beta (C/EBPβ), perilipin 2 (PLIN2), and fatty acid binding protein 3 (FABP3), whereas miR-27a downregulation increased PPARG, C/EBPβ, FABP3, and CCAAT enhancer binding protein alpha (C/EBPα) mRNA expression. Furthermore, Western blot analysis revealed the protein level of PPARG in miR-27a mimic and inhibitor transfection groups to be consistent with the mRNA expression response. Moreover, luciferase reporter assays verified that PPARG was the direct target of miR-27a. In summary, these results indicate that miR-27a has the ability to control TAG synthesis in BMEC via targeting PPARG, suggesting that miR-27a could potentially be used to improve beneficial milk components in dairy cows.

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.

Milk fat is a main nutritional component of milk, and it has become one of the important traits of dairy cow breeding. Recently, there is increasing evidence that microRNAs (miRNA) play significant roles in the process of milk fat synthesis in the mammary gland. Primary bovine mammary epithelial cells (BMEC) were harvested from midlactation cows and cultured in DMEM/F-12 medium with 10% fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL bovine insulin, 1 µg/mL hydrocortisone, and 2 µg/mL bovine prolactin. We found that miR-34b mimic transfection in BMEC reduced the content of intracellular triacylglycerol (TAG) and lipid droplet accumulation via triacylglycerol assay and Oil Red O staining; meanwhile, overexpression of miR-34b inhibited mRNA expression of lipid metabolism-related genes such as peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), fatty acid binding protein 4 (FABP4), and CCAAT enhancer binding protein alpha (C/EBPα). Whereas miR-34b inhibitor resulted in completely opposite results. Furthermore, q-PCR and western blot analysis revealed the mRNA and protein expression levels of DCP1A were downregulated in miR-34b mimic transfection group and upregulated in miR-34b inhibitor group. Moreover, luciferase reporter assays verified that DCP1A was the direct target of miR-34b and DCP1A gene silencing in BMEC-inhibited TAG accumulation and suppressed lipid droplet formation. In conclusion, these findings revealed a novel miR-34b–DCP1A axis that has a significant role in regulating milk fat synthesis and suggested that miR-34b may be used to improve the beneficial ingredients in milk.

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.

Castration increases fat deposition, improving beef quality in cattle. Here, the steer group exhibited a significantly higher intramuscular fat (IMF) content than the bull group. To determine the potential roles of microRNAs (miRNAs) in castration-induced fat deposition, differential expression patterns of miRNA in liver tissue were investigated in bulls and steers. A total of 7,827,294 clean reads were obtained from the bull liver library, and 8,312,483 were obtained from the steer liver library; 452 conserved bovine miRNAs and 20 novel miRNAs were identified. The results showed that the expression profiles of miRNA in liver tissue were changed by castration, and 12 miRNAs that were differentially expressed between bulls and steers were identified. Their target genes were majorly involved in the metabolic, PI3K-Akt, and MAPK signaling pathways. Furthermore, six differentially expressed miRNAs were validated by quantitative real-time PCR, and luciferase reporter assays verified that calcium-sensing receptor (CASR) was the direct target of miR-27a-5p. Meantime, we found that the expression level of CASR was significantly higher in steers than in bulls, and revealed that CASR gene silencing in bovine hepatocytes significantly inhibited triacylglycerol (TAG) accumulation and reduced secretion of very low density lipoprotein (VLDL). These results obtained in the liver indicate that miR-27a-5p may increase fat deposition partly by targeting CASR in steers.