miR-380-3p promotes β-casein expression by targeting αS1-casein in goat mammary epithelial cells

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.


INTRODUCTION
Milk protein is a dietary protein source that supplies essential amino acids and bioactive peptides for humans, mainly composed of caseins and whey proteins.Caseins account for about 80% of the total milk protein, and are divided into α S1 -, α S2 -, β-and κ-casein.Despite its benefits, milk protein also carries a risk of food allergies for many people, especially for infants and children [1].The global prevalence of milk protein allergy was reported to be as high as 2% to 7.5% [2].Among the numerous components of milk protein, α S1 -casein is most prone to cause allergic reactions, and approximately 65% of patients with milk allergies are caused by α S1 -casein [3].Goat milk contains a variable content of α S1 -casein (4% to 26% of milk protein) compared to cow milk (36% to 40%) [4].Therefore, the method of regulating α S1 -casein synthesis to inhibit the allergic potential of milk protein is necessary to be elucidated.
MicroRNA (miRNA) is a class of small non-coding RNA of about 22 nucleotides in length, which regulate mRNA expression by targeting the 3'-untranslated region (3'UTR) of gene transcripts.The pairing between miRNA seed site sequence (2nd to 8th nucleotides at the 5' terminal) and target genes is considered a key feature for mammalian miRNA to recognize its targets.The abundance of miR-380-3p is involved in the prevention and treatment of a variety of human diseases.miR-380-3p participates in intracorporal neurodegeneration induced by neurotoxicants via specificity protein 3 signaling pathway [5].High expression of miR-380-3p inhibits cell proliferation and invasion by targeting oncogene Kruppel-like factor 4 in triple negative breast cancer cells [6].miR-380-3p increases the anti-proliferative and pro-apoptotic effects of doxorubicin in multiple models of high-risk neuroblastoma [7].
To address the milk allergy caused by α S1 -casein in ruminants, it is urgent to investigate the regulatory mechanism of miRNAs on α S1 -casein synthesis.Although multiple studies have revealed that miR-380-3p plays the pivotal role in a variety of biological processes, whether it could modulate ruminant milk protein expression is unknown.α S1 -Casein gene was targeted by miR-380-3p in dairy goats or cows via TargetScan database (http://www.targetscan.org/)prediction.Hence, the aim of our study was to explore the molecular mechanism by which miR-380-3p controls milk protein synthesis through the target gene in goat mammary epithelial cells (GMEC).In this study, we revealed that miR-380-3p reduced α S1 -casein synthesis and enhanced β-casein abundance in GMEC, which supplies a novel approach to weaken milk allergy potential and promote milk nutritional quality in ruminants.

Animal care
The animal study protocol was approved by the Institutional Animal Care and Use Committee, Northwest A&F University, YangLing, China (protocol code .

Statistical analysis
All experiments were repeated for at least for 3 biological replicates.Data were shown as mean±standard error of the mean and analyzed by SPSS 20.0 software (IBM, Chicago, IL, USA).T-test was carried out with two group data, and one-way analysis of variance with Tukey test was carried out for multiple comparison.The difference was significant when p<0.05 (* p<0.05, ** p<0.01).
Further, we attempted to identify the binding of STAT5 to β-casein promoter by ChIP analysis.Compared with rabbit IgG control group, STAT5 forcefully bound to the specific site of β-casein promoter region in p-STAT5 group (p<0.01),but STAT5 inhibitor highly attenuated the combination of STAT5 and β-casein promoter region (p<0.01; Figure 5E).Subsequently, we discovered that the binding efficiency of STAT5 and β-casein promoter was significantly enhanced by miR-380-3p mimic treatment (p<0.05; Figure 5F).These data declared that STAT5 activated β-casein transcription and that the modulation of STAT5 on β-casein promoter was increased by miR-380-3p overexpression.Overall, our study revealed that miR-380-3p inhibits α S1 -casein expression by targeting α S1 -casein, and indirectly promotes β-casein transcription via α S1 -casein/STAT5 axis in GMEC (Figure 6).

DISCUSSION
Understanding the regulatory mechanism of miRNA in physiological progresses could provide the theoretical basis for its application in animals.Previous studies have shown miRNAs modulate the synthesis process of milk proteins.miR-424-5p targeted 3'UTR of β-casein gene by prolactin receptor signaling pathway in murine mammary glands [10].miR-2904 positively regulated milk protein synthesis via Janus kinase 2 (JAK2)/STAT5 signaling pathway by the functional network analysis of Chinese Holstein cows [11].In bovine mammary epithelial cells (BMEC), miR-139 inhibited β-casein expression through targeting growth hormone receptor and insulin-like growth factor receptor [12].miR-27a negatively regulated milk protein and lactose contents through targeting mitogen-activated protein kinase 14 that had a positive effect on milk protein expression in sheep [13].In GMEC, miR-8516 promoted the secretion of α S1 -casein and β-casein by activating the activity of JAK2/STAT5 [14].Although these studies indicate the miRNA regulation of  milk protein synthesis, research on miRNA-targeted α S1 -casein expression are still insufficient.According to the TargetScan software prediction, miR-380-3p targets 3'UTR region of goat α S1 -casein gene.A dual-luciferase experiment was carried out to identify the targeted relationship of miR-380-3p to α S1 -casein gene.More importantly, miR-380-3p decreased the expression of α S1 -casein gene and α S1 -casein in dairy goats, which contributed to low allergy potential in goat milk.miR-380-3p is involved in multiple biological progresses, such as cell proliferation, apoptosis, inflammation and melanogenesis.miR-380-3p targeted sex determining region Ybox 6 to regulate melanogenesis by affecting beta-catenin and microphthalmia-associated transcription factor expression in melanocytes [15].High expression of miR-380-3p increased cell inflammation by promoting tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-18 levels in spinal cord jury model rats [16].Overexpression of miR-380-3p activated inflammation, oxidative stress, and apoptosis by targeting suppressor of cytokine signaling 6 in retinal epithelial cells with diabetic retinopathy [17].In mouse neuroblastoma cells, miR-380-3p inhibited cell proliferation and increased cell apoptosis rate through blocking the translation of transcription factor specificity protein-3 [18].On the contrary, miR-380-3p, acted as an oncogenic miRNA, promoted cell proliferation, epithelial-mesenchymal transition, and tumorigenesis in pancreatic cancer cells [19].Despite the potent features of miR-380-3p, its regulatory role in milk protein expression is still unknown.In our research on dairy goats, we uncovered that miR-380-3p decreased α S1 -casein expression and enhanced β-casein abundance by targeting 3'UTR of α S1 -casein gene.We expected that there was a correlation between α S1 -casein and β-casein abundance regulated by miR-380-3p, which should be investigated in more detail in GMEC.
Multiple studies have shown the association between the expression of different milk protein components.In mouse mammary epithelial cells, lipoteichoic acid treatment increased the intracellular and secreted α S1 -casein accompanied by a decrease in β-casein content [20].Compared with wildtype dairy cows, the contents of α S1 -casein, β-casein, κ-casein and α-lactalbumin were all increased in β-lactoglobulin silenced cows [21].On the contrast, the contents of α-casein and α-lactalbumin in milk from β-casein and κ-casein transgenic cows were lower than non-transgenic cows [22].There was a negative correlation between α S1 -casein and β-casein gene expression in BMEC treated with camellia seed oil [23].In mammary gland of lactation goats, α S1 -casein gene expression was negatively correlated with β-lactoglobulin mRNA level [24].More importantly, our previous study found that α S1 -casein gene negatively regulated STAT5a phosphorylation and β-casein expression in GMEC [8].In this research on GMEC, we carried out rescue assays to identify whether miR-380-3p up-regulated p-STAT5a and β-casein abundance by targeting α S1 -casein gene.miR-380-3p mimic rescued the inhibition of α S1 -casein gene on p-STAT5a and β-casein expression, while miR-380-3p inhibitor moderated the impact of α S1 -casein gene knockdown on p-STAT5a and β-casein abundance.These rescue assays reveal that miR-380-3p indirectly increases STAT5 activity and β-casein expression by inhibiting α S1 -casein expression.The present results are supported by our previous study that α S1 -casein downregulated STAT5 activity and β-casein expression.
Previous studies have shown STAT5 is a primary transcriptional factor for milk production in mammals.STAT5 binds to milk protein gene promoters by identifying the specific binding motif.In STAT5 knockout mouse model, there was a significant reduction in the content of α-lactalbumin and β-lactoglobulin in milk [25].Curcumin and basolateral lactose exposure suppressed intracellular and secreted casein production by STAT5 inactivation in mouse mammary epithelial cells [26].In BMEC, beta-sitosterol promoted β-casein synthesis through the activation of STAT5 [27]; biochanin A declined β-casein synthesis and secretion by inhibiting STAT5 activity [28].Milk protein expression was markedly decreased when STAT5a activity was blocked by suppressor of cytokine signaling 3 in dairy cows [29].Further, phosphorylated STAT5a bound to β-casein promoter region and increased β-casein synthesis in immortalized GMEC [30].Consistent with previous studies, luciferase experiments have shown that phosphorylated STAT5 promoted β-casein gene transcription as well in the present study.More importantly, ChIP experiments were carried out to demonstrate the direct binding of STAT5 to β-casein gene promoter, which could be activated by miR-380-3p mimic.Therefore, miR-380-3p indirectly promotes β-casein transcription through α S1 -casein/STAT5a regulation axis.Additionally, α S1 -casein gene could be targeted by miR-380-3p both in dairy goats and cows via TargetScan database prediction.In this study, we verified that miR-380-3p targets α S1 -casein gene and promotes STAT5 activity and β-casein expression by inhibiting α S1 -casein expression in dairy goats.However, whether miR-380-3p could target α S1 -casein gene and play similar regulatory roles needs to be further studied in dairy cows.

CONCLUSION
miR-380-3p targets 3'UTR of goat α S1 -casein gene and inhibits α S1 -casein expression which is associated with higher rates of milk allergy for humans.miR-380-3p promotes β-casein expression through targeting α S1 -casein gene.Moreover, miR-380-3p increases β-casein transcription by the binding of STAT5 to β-casein gene promoter region.In summary, our study is helpful to conclude the regulatory role of miR-380-3p on milk protein synthesis and supplies

Table 1 .
Primers used for quantitative real-time polymerase chain reaction of genes and promoter region UXT, ubiquitously expressed transcript; RPS9, ribosomal protein S9.