Compared with other milk proteins, α_S1-casein (encoded by CSN1S1) is associated with higher rates of allergies in humans. Although some studies have revealed that CSN1S1 affects a variety of cellular physiological processes such as immune response and proliferation in various cells, whether CSN1S1 regulates other milk proteins in ruminants is not known. In this study, we observed a negative Pearson correlation between the contents of α_S1-casein and β-casein in goat milk. Thus, we used isolated primary goat mammary epithelial cells along with adenoviral infection or small interference RNA to alter abundance of CSN1S1 and examine its role in milk protein synthesis regulation. Overexpressing CSN1S1 through adenoviral transfection decreased β-casein mRNA (CSN2) and protein abundance, whereas interference of CSN1S1 resulted in a significant increase in β-casein abundance. CSN1S1 reduced phosphorylation level of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5a (STAT5a). The transcription factor STAT5a activates CSN2 transcription by binding with its promoter region and then promotes β-casein synthesis. Furthermore, CSN1S1 inhibited CSN2 promoter activity and β-casein synthesis by modulating the JAK2/STAT5a signaling pathway. No changes in abundance were detected for α_S2-casein (CSN1S2), κ-casein (CSN3) and major whey proteins (LALBA, BLG). Overall, our results underscored a mechanism whereby CSN1S1 inhibits β-casein synthesis via inhibition of STAT5a. The data suggested that knocking down CSN1S1 not only reduced the content of α_S1-casein, but also increased the abundance of major milk proteins including β-casein. Thus, the present study provides a theoretical basis for manipulating α_S1-casein mRNA to improve quality of ruminant milk for human consumption.

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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.

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.

Objective: αS1-Casein is more closely associated with milk allergic reaction than other milk protein components. microRNA (miRNA) is a class of small non-coding RNAs that modulate multiple biological progresses by the target gene. However, the post-transcriptional regulation of αS1-casein expression by miRNA in ruminants remains unclear. This study aims to explore the regulatory roles of miR-380-3p on αS1-casein synthesis in goat mammary epithelial cells (GMEC).Methods: αS1-Casein gene and miR-380-3p expression was measured in dairy goat mammary gland by quantitative real-time polymerase chain reaction (qRT-PCR). miR-380-3p overexpression and knockdown were performed by miR-380-3p mimic or inhibitor in GMEC. The effect of miR-380-3p on αS1-casein synthesis was detected by qRT-PCR, western blot, luciferase and chromatin immunoprecipitation assays in GMEC.Results: Compared with middle-lactation period, αS1-casein gene expression is increased, while miR-380-3p expression is decreased during peak-lactation of dairy goats. miR-380-3p reduces αS1-casein abundance by targeting the 3’-untranslated region (3’UTR) of αS1-casein mRNA in GMEC. miR-380-3p enhances β-casein expression and signal transducer and activator of transcription 5a (STAT5a) activity. Moreover, miR-380-3p promotes β-casein abundance through target gene αS1-casein, and activates β-casein transcription by enhancing the binding of STAT5 to β-casein gene promoter region.Conclusion: miR-380-3p decreases αS1-casein expression and increases β-casein expression by targeting αS1-casein in GMEC, which supplies a novel strategy for reducing milk allergic potential and building up milk quality in ruminants.