Malonyl-CoA decarboxylase (MCD) is a major regulator of fatty acid oxidation catalyzing the decarboxylation of malonyl coenzyme A (malonyl-CoA). Although its involvement in human diseases has been well studied, its role in intramuscular fat (IMF) deposition remains unknown. In this present study, 1726 bp of MCD cDNA was cloned (OM937122) from goat liver, including 5′UTR of 27 bp, 3′UTR of 199 bp, and CDS of 1500 bp, encoding 499 amino acids. In this present study, although the overexpression of MCD increased the mRNA expression of FASN and DGAT2, the expression of ATGL and ACOX1 was also activated significantly and resulted in a decrease in cellular lipid deposition in goat intramuscular preadipocytes. Meanwhile, the silencing of MCD increased the cellular lipid deposition and was accompanied by the expression activation of DGAT2 and the expression suppression of ATGL and HSL, despite the expression suppression of genes related to fatty acid synthesis, including ACC and FASN. However, the expression of DGAT1 was not affected significantly (p > 0.05) by the expression alteration of MCD in this present study. Furthermore, 2025 bp of MCD promoter was obtained and predicted to be regulated by C/EBPα, SP1, SREBP1, and PPARG. In summary, although different pathways may respond to the expression alteration of MCD, the expression of MCD was negatively correlated with the cellular lipid deposition in goat intramuscular preadipocytes. These data may be beneficial for enhancing our understanding of the regulation of IMF deposition in goats.

MicroRNA (miRNA)-130b, as a regulator of lipid metabolism in adipose and mammary gland tissues, is actively involved in lipogenesis, but its endogenous role in fatty acid synthesis remains unclear. Here, we aimed to explore the function and underlying mechanism of miR-130b in fatty acid synthesis using the CRISPR/Cas9 system in primary goat mammary epithelial cells (GMEC). A single clone with deletion of 43 nucleotides showed a significant decrease in miR-130b-5p and miR-130b-3p abundances and an increase of target genes PGC1α and PPARG. In addition, knockout of miR-130b promoted triacylglycerol (TAG) and cholesterol accumulation, and decreased the proportion of monounsaturated fatty acids (MUFA) C16:1, C18:1 and polyunsaturated fatty acids (PUFA) C18:2, C20:3, C20:4, C20:5, C22:6. Similarly, the abundance of fatty acid synthesis genes ACACA and FASN and transcription regulators SREBP1c and SREBP2 was elevated. Subsequently, interference with PPARG instead of PGC1α in knockout cells restored the effect of miR-130b knockout, suggesting that PPARG is responsible for miR-130b regulating fatty acid synthesis. Moreover, disrupting PPARG inhibits PGC1α transcription and translation. These results reveal that miR-130b directly targets the PPARG–PGC1α axis, to inhibit fatty acid synthesis in GMEC. In conclusion, miR-130b could be a potential molecular regulator for improving the beneficial fatty acids content in goat milk.

α_S1-Casein (encoded by the CSN1S1 gene) is associated with food allergy more than other milk protein components. Milk allergy caused by α_S1-casein is derived from cow milk, goat milk and other ruminant milk. However, little is known about the transcription regulation of α_S1-casein synthesis in dairy goats. This study aimed to investigate the regulatory roles of signal transducer and activator of transcription 5 (STAT5) on α_S1-casein in goat mammary epithelial cells (GMEC). Deletion analysis showed that the core promoter region of CSN1S1 was located at −110 to −18 bp upstream of transcription start site, which contained two putative STAT5 binding sites (gamma-interferon activation site, GAS). Overexpression of STAT5a gene upregulated the mRNA level and the promoter activity of the CSN1S1 gene, and STAT5 inhibitor decreased phosphorylated STAT5 in the nucleus and CSN1S1 transcription activity. Further, GAS site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays revealed that GAS1 and GAS2 sites in the CSN1S1 promoter core region were binding sites of STAT5. Taken together, STAT5 directly regulates CSN1S1 transcription by GAS1 and GAS2 sites in GMEC, and the mutation of STAT5 binding sites could downregulate CSN1S1 expression and decrease α_S1-casein synthesis, which provide the novel strategy for reducing the allergic potential of goat milk and improving milk quality in ruminants.

MicroRNA-26 (miR-26a and miR-26b) plays a critical role in lipid metabolism, but its endogenous regulatory mechanism in fatty acid metabolism is not clear in goat mammary epithelial cells (GMECs). GMECs with the simultaneous knockout of miR-26a and miR-26b were obtained using the CRISPR/Cas9 system with four sgRNAs. In knockout GMECs, the contents of triglyceride, cholesterol, lipid droplets, and unsaturated fatty acid (UFA) were significantly reduced, and the expression of genes related to fatty acid metabolism was decreased, but the expression level of miR-26 target insulin-induced gene 1 (INSIG1) was significantly increased. Interestingly, the content of UFA in miR-26a and miR-26b simultaneous knockout GMECs was significantly lower than that in wild-type GMECs and miR-26a- and miR-26b-alone knockout cells. After decreasing INSIG1 expression in knockout cells, the contents of triglycerides, cholesterol, lipid droplets, and UFAs were restored, respectively. Our studies demonstrate that the knockout of miR-26a/b suppressed fatty acid desaturation by upregulating the target INSIG1. This provides reference methods and data for studying the functions of miRNA families and using miRNAs to regulate mammary fatty acid synthesis.

In nonruminants, microRNA (miRNA)-24 plays an important role in lipid metabolism in adipose tissue and the liver. Although the abundance of miR-24 in ruminant mammary glands is the highest during peak lactation, its potential role in regulating the synthesis and secretion of fat into milk is unclear. This study aimed to identify the function of miR-24 in these processes using CRISPR/Cas9 technology in primary goat mammary epithelial cells (GMEC). A single clone containing a 66-nucleotide deletion between two sgRNAs mediating double-strand break (DSB) sites was obtained. The abundance of miR-24-3p and miR-24-5p encoded by the deleted sequence was decreased, whereas the target genes INSIG1 and FASN increased. In addition, miR-24 knockout reduced the gene abundance of genes associated with fatty acid and TAG synthesis and transcription regulator. Similarly, the content of cholesterol and monounsaturated fatty acid (MUFA) C18:1 decreased, whereas that of polyunsaturated fatty acids (PUFA) C18:2, C20:3, C20:4 and C20:5 increased. Subsequently, knocking down of INSIG1 but not FASN reversed the effect of miR-24 knockout, indicating that miR-24 modulated cholesterol and fatty acid synthesis mainly by targeting INSIG1. Overall, the present in vitro data demonstrated a critical role for miR-24 in regulating lipid and fatty acid synthesis and highlighted the possibility of manipulating milk components in dairy goats.

Meat quality in goats is partly determined by the intramuscular fat (IMF) content, which is associated with the proliferation and differentiation of intramuscular preadipocytes. Emerging studies have suggested that miRNA plays a crucial role in adipocyte proliferation and differentiation. In our recent study, we observed the expression variations in miR-196a in the longissimus dorsi muscle of Jianzhou goats at different ages. However, the specific function and underlying mechanism of miR-196a in IMF deposition are still unclear. This study demonstrated that miR-196a significantly enhanced adipogenesis and apoptosis and reduced the proliferation of preadipocytes. Subsequently, RNA-seq was employed to determine genes regulated by miR-196a, and 677 differentially expressed genes were detected after miR-196a overexpression. The PI3K-Akt pathway was identified as activated in miR-196a regulating intramuscular adipogenesis via Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and further verified via Western blot and rescue assays. Lastly, using RT-qPCR, Western blot, dual-luciferase, and rescue assays, we found that miR-196a promoted adipogenesis and suppressed the proliferation of intramuscular preadipocytes by the downregulation of MAP3K1. In summary, these results suggest that miR-196a regulates IMF deposition by targeting MAP3K1 and activating the PI3K-Akt pathway and provide a theoretical foundation for improving goat meat quality through molecular breeding.