MiR-486 Regulates Lactation and Targets the PTEN Gene in Cow Mammary Glands

Mammary gland development is controlled by several genes. Although miRNAs have been implicated in mammary gland function, the mechanism by which miR-486 regulates mammary gland development and lactation remains unclear. We investigated miR-486 expression in cow mammary gland using qRT-PCR and ISH and show that miR-486 expression was higher during the high-quality lactation period. We found that miR-486 targets phosphoinositide signaling in the cow mammary gland by directly downregulating PTEN gene expression and by altering the expression of downstream genes that are important for the function of the mammary gland, such as AKT, mTOR. We analyzed the effect of β-casein, lactose and triglyceride secretion in bovine mammary gland epithelial cells (BMECs) transfected by an inhibitor and by mimics of miR-486. Our results identify miR-486 as a downstream regulator of PTEN that is required for the development of the cow mammary gland.


Introduction
Bovine mammary glands arise from the ectoderm during embryonic development and continue to develop postnatally through puberty, pregnancy, lactation, and subsequent involution. Most developmental and functional differentiation in the mammary gland occurs after the birth of offspring [1]. During lactation, the mammary gland secretes milk, which provides nearly all the nutrient requirements of the newborn offspring during the transition from pregnancy to lactation [2]. The gland develops primarily postnatally, and its development is mainly controlled by steroids, peptide hormones, and cell matrix interactions during different stages. Several pathways have been shown to modulate the progression of mammary gland development. Additionally, more than 100 genes have been shown to modulate various aspects of mammary physiology, from the formation of the fetus to remodeling of the gland during involution [2][3]. MicroRNAs (miRNAs) have also been shown to regulate cell processes, and many miRNAs are involved in mammary gland development and tumorigenesis [4].
Due to the unique developmental features found during distinct stages of lactation, the mammary gland represents an important model for use in studies to elucidate signaling related to cell cycle progression, survival, proliferation, differentiation, and cell death. Despite the relatively recent recognition of miRNAs as key regulators of cellular function, little research has groups by developmental stage: the lactation with high quality milk stage (milk yield 30.6±0.78 kg/d, milk protein >3.0%, and milk fat >3.5%; n = 3), the lactation with poor quality milk stage (milk yield 30.6±0.78 kg/d, milk protein <3.0%, and milk fat <3.5%; n = 3), and the pregnancy stage (n = 3). All of the cows were fed with standard foodstuffs consisting of 30% roughage and 70% concentrate. The cows were slaughtered by exsanguinations, and their mammary tissue was aseptically excised 5 cm from the base of the healthy breast and 3 cm from the midline that divides the core of the secretory gland tissue. After removing the connective tissue, the remaining tissue was cut into small blocks with a thickness of 1 cm. The mammary tissue samples were immediately frozen in liquid nitrogen and stored at-80°C for later analysis. All animal experiments were approved by the Institutional Animal Care and Use Ethics Committee of Northeast Agricultural University and conducted in accordance with the Guidelines for Experimental Animals from the Ministry of Science and Technology (Beijing, China).
The indicated cells were transfected with 50 nmol miR-486 mimics, inhibitors, or the corresponding negative control constructs (miR-NC, Anti-NC) (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions, respectively. Twenty-four hours after transfection, the cells were treated with cell differentiation medium.

Small RNA sequencing
We extracted total RNA from mammary gland tissues at 3 different developmental stages using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA samples were sent to the Beijing Genomics Institute (Shenzhen, Guangdong, China), where a small RNA library was constructed and sequenced using a Genome Analyzer (Illumina, San Diego, USA). All of the data were stored in the Short Archive section of the NCBI GEO under accession number GSE57991. The sequencing data were first filtered into mRNA using the Rfam 10.1 and Genebank databases and then mapped to miRBase 18. The mapped data were used to identify significant differences in miRNA expression [18].

Luciferase activity assay
HEK-293 cells were cultured in 24-well plates, and each plate was transfected with 80 ng of pGL3/PTEN vector or pGL3/PTEN/mut vector containing firefly luciferase and 4 ng of the pRL-TK vector (Promega Madison, WI, USA) containing 4 pmol miR-486 mimics or negative control. The cells were transfected using Lipofectamine 2000 (Invitrogen). After 48 h of transfection, relative luciferase activity was calculated by normalizing the firefly luminescence to the renilla luminescence using the Dual-Luciferase Reporter Assay (Promega Madison, WI, USA) according to the manufacturer's instructions. The experiments were performed in triplicate.

Quantitative polymerase chain reaction (qPCR) assays
After the transfection of the cells with the miR-486 mimics/inhibitors, total RNA was purified using Trizol reagent (Invitrogen) following the manufacturer's instructions. cDNA (complementary DNA) was synthesized using the PrimerScript RT-PCR kit (TaKaRa) according to the manufacturer's instructions.
The expression of miR-486 and the 5S internal control gene were quantified using real-time PCR quantification and the Hairpin-it miRNAs qPCR Quantitation Kit (GenePharma, Shanghai, China) according to the manufacturer's protocol. Specific primers for miR-486 and 5S were designed by GenePharma. The expression of these genes was analyzed using a 7300 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).
The expression of PTEN, AKT, mTOR, and β-actin was quantified using Real-Time-PCR quantification and SYBR Premix Ex Taq II (TaKaRa) according to the manufacturer's instructions. β-actin was used as a loading control. The primers that were used for qRT-PCR are listed in Table 1. All qPCR reactions were performed in triplicate.

In Situ hybridization
In situ hybridization (ISH) experiments with 5-μm paraffin sections of bovine mammary gland tissues were performed using a DIG-labeled locked nucleic acid probe against miR-486 (5-CTCGGGGCAGCTCAGTACAGGA-3), used scramble (Exiqon, USA) as the negative control probe. Tissues were dewaxed, postfixed in 4% paraformaldehyde (PFA) for 1 h, and digested in proteinase K solution (5 μg/ml) at 37°C for 5 min. The slides were pre-hybridized at 37°C for 1 h in 50% formamide. The LNA probe was diluted with ISH buffer (1.5 pM), and the probe was denatured at 95°C for 8 min. Hybridization was performed overnight at 55°C, followed by post-hybridization washes in 5×SSC and 0.2×SSC at 60°C. After exposure for 10 min to 3% H 2 O 2 , the slides were incubated with a mouse monoclonal antibody against digoxin (Abcam Technology, Cambridge, MA, USA) at 37°C for 30 min and then incubated with an HRPconjugated goat-anti-murine secondary antibody (Abcam). After antibody treatment, the sections were incubated with 0.04% DAB + 0.03% H 2 O 2 to develop the color for 30 min, after which the sections were counterstained slightly with Mayer's hematoxylin. An Olympus CX31 microscope was used.

Western blot analysis
After the overexpression and inhibition of miR-486, the cell lysates were separated on a 10% SDS-PAGE gel (30 μg protein per sample), and the proteins were then transferred onto nitrocellulose membranes (Bio-Rad, Shanghai, China). Nonspecific binding sites were blocked using a 5% skim milk solution for 1 h at 37°C, and the membranes were then incubated with a rabbit polyclonal antibody against PTEN (Santa Cruz Biotechnology Inc., Dallas, TX, USA), AKT, phospho-AKT (Ser473), MTOR (Cell Signaling Technology, Beverly, MA, USA), or phospho-MTOR (S2448) or a murine polyclonal antibody against β-actin (Santa Cruz Biotechnology Inc.) overnight at 4°C; this was followed by incubation with goat-anti-rabbit or goatanti-murine secondary antibodies conjugated to HRP (Zhongshan-Bio, Beijing, China), and the proteins were visualized using Super ECL Plus (ApplyGEN, Beijing, China). β-actin was used as a loading control.

Cell cycle analysis
Cell cycle analysis was performed using flow cytometry (FCM) as follows: harvested cells were washed with cold PBS and fixed with 70% ethanol overnight at 4°C, and washed with cold PBS. The cells were then incubated with PI (50 μg/ml) and mixed with 2 μl/ml TritonX-100 for 20 min at room temperature in the dark. The cells were then suspended in 500 μl PBS. Samples were analyzed using flow cytometry on a Cytomics TMFC500 flow cytometer, and the data were analyzed using Mod Fit LT 3.2 software (Verity Software House, USA). The FCM analysis was carried out in triplicate.

Cell Proliferation
Cell proliferation was determined using the colorimetric water-soluble tetrazolium salt (WST-8) assay using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Japan) according to the manufacturer's instructions. After 24 h of transfection with miR-486 mimics/inhibitors, BMECs were seeded onto a 96-well plate (4×10 4 cells per 100 μl per well), and 10 μl of CCK-8 solution was added to each well. Cells were incubated for 2 h, and cell proliferation was documented. Absorbance was measured at 450 nm using a microplate reader with a reference wavelength of 650 nm. Averages of 6 replicates were analyzed, and statistical analysis was performed using the t-test.

Cell proliferation assay
BMECs were seeded in glass-bottomed cell culture dishes. After 48 h of transfection, cell proliferation was quantified based on the incorporation of 5-ethynyl-2-deoxyuridine (EdU) into

Statistical analysis
The results were analyzed using SPSS 17.0 statistics software (Chicago, IL, USA). All results were expressed as the means ± standard deviation (SD) of separate experiments (n3). P values less than 0.05 were considered significant.

Expression of miR-486 in bovine mammary gland tissues with different milk qualities
To examine whether miR-486 expression depends on the stage of mammary development specifically during the high-milk-quality lactation period (H) (Fig. 1A-1D), the low-milk-quality lactation period (L) (Fig. 1E-1H), and the pregnant period (P) (Fig. 1I-1L), we used in situ hybridization to evaluate the expression of miR-486 in bovine glandular tissue and adipose tissue (Fig. 1A). In situ hybridization using a DIG-labeled locked nucleic acid probe against miR-486 revealed that miR-486 was primarily expressed in bovine mammary glandular epithelium tissue ( Fig. 1A: H-a, L-a, P-a) and was not expressed in bovine mammary adipose tissue ( Fig. 1A: H-c, L-c, P-c). Additionally, to determine if miR-486 expression was different between the high-milk quality and low-milk quality lactation periods we used a small RNA sequencing approach developed by our laboratory. And we found that the glandular expression of miR-486 was higher in high milk quality cows than in low milk quality cows (P<0.01) and pregnant cows (P<0.01). These results are shown in Fig. 1B. Furthermore, qRT-PCR analysis showed that the expression of miR-486 was higher in the H group than in the L (P<0.01) and P groups (P<0.01) (Fig. 1C).

MiR-486 targets the PTEN gene
MiR-486 is conserved in mammals and is the sole miRNA with no known family members [14]. Using the prediction software TargetScan6.2, we found that miR-486 target sites are present at nt 725-732 and nt 3,183-3,190 of the PTEN 3 0 UTR; these two sites are conserved in mammals. The 3 0 UTR of wild-type PTEN was linked to pGL3 Luciferase Reporter Vectors (Promega) and examined in HEK-293 cells that had been transfected with miR-486 mimics or a negative control. A significant decrease in reporter activity was observed when using the miR-486 mimics compared to the negative control. Transfection with miR-486 mimics had little effect on the luciferase activity of the mutant 3 0 UTR-reporter of PTEN compared with the negative control. Moreover, the luciferase signal of the pGL3-control vector with miR-486 was similar to that obtained with the negative control as shown in Fig. 2.

MiR-486 inversely correlates with PTEN in BMECs
We found that the expression of miR-486 increased after treatment with miR-486 mimics (Fig. 3 A, P<0.0001) and decreased after treatment with miR-486 inhibitors (Fig. 3 B,  P<0.0001). To investigate whether miR-486 affects the regulation of the PTEN in mammary epithelial cells, we measured the relative expression of PTEN using qRT-PCR and western blotting. We found that the level of miR-486 changed concomitantly with the mRNA level of PTEN and that miR-486 inhibited the expression of PTEN (Fig. 3 C, Fig. 3 E, P<0.0001). Transfecting BMECs with a miR-486 inhibitor increased the expression of PTEN (Fig. 3 D, P<0.01; Fig. 3F, P<0.0001). We assessed the expression of PTEN after treatment with miR-486 mimics/inhibitors in BMECs using immunohistochemistry. We observed the expression of PTEN both in the cell nuclei and cytoplasm of mammary gland tissues (Fig. 3 G). A high expression of miR-486 repressed the expression of PTEN in mammary epithelium cell nuclei. However, a reduced expression of miR-486 stimulated PTEN expression in mammary epithelial cell nuclei. Thus, the data above suggest that miR-486 negatively affects PTEN.

MiR-486 increases the expression of AKT and MTOR in bovine mammary epithelial cells
MiR-486 significantly repressed PTEN expression in cell culture and induced AKT and MTOR expression in BMECs. We observed that, compared to a negative control, the high-level expression of miR-486 stimulated the expression of AKT and MTOR (Fig. 4 A, P<0.0001). Moreover, the knockdown miR-486 repressed the expression of AKT and MTOR (Fig. 4 B, P<0.0001). The relative protein expression of AKT, phospho-AKT (p-AKT), MTOR, and phospho-MTOR (p-MTOR) increased as the expression of miR-486 was increased (Fig. 4 C, P<0.0001). However, decreasing the levels of miR-486 reduced the relative protein expression of AKT, p-AKT, MTOR, and p-MTOR (Fig. 4 D; P<0.0001, P<0.0001, P<0.01, respectively).  To assess whether miR-486 affects the proliferation of bovine mammary epithelial cells, these cells were transfected with miR-486 mimics/inhibitor and analyzed using CCK-8 assays after 48 h. We measured the proliferation rate of the miR-486 mimics group and the miR-486 inhibitor group; miR-486 mimics increased the ability of the cells to proliferate, and the miR-486 inhibitor suppressed the ability of the cells to proliferate (Fig. 5 A). Additionally, we used the EdU incorporation assay to determine the role of miR-486 in the proliferation of BMECs. The percentage of Edu-positive cells was significantly higher in the miR-486 mimic-treated cells than in the negative control group (P<0.05). Additionally, the percentage of Edu-positive cells was lower in the miR-486 inhibitor group than in the negative control group (Anti-NC) (Fig. 5 B, P<0.05).
Next, BMECs transfected with miR-486 mimics, inhibitor, or negative control were examined for changes in the cell cycle using flow cytometry analysis (Fig. 6). Transfection with miR-486 mimics resulted in an increased population of cells in the S phase and a decreased population of cells in G0/G1 compared to transfection with the negative control ( Fig. 6 A, P<0.05). Furthermore, miR-486 inhibitors caused the opposite effect compared to the negative control (Anti-NC), (Fig. 6 B, P<0.05). The data above indicate that miR-486 is a negative modulator of the G1 to S transition. β-casein levels, lactose secretion, and triglyceride levels are upregulated by miR-486 Mammary epithelial cells synthesize and secrete proteins, lactose, and lipids. Thus, we examined the supernatant for changes in the levels these key components. The overexpression of miR-486 increased the concentrations of triglycerides, β-casein and lactose (Fig. 7 A, 7 C, and 7 E, P<0.0001; P<0.0001; P<0.001); conversely, inhibition of miR-486 suppressed the concentrations of triglycerides, β-casein and lactose (Fig. 7 B, 7 D, and 7 F, P<0.0001).

Discussion
Currently, many miRNAs have been identified using algorithmic and experimental methods in animals, plants, and viruses. Many miRNAs have been found to play roles in cell differentiation, proliferation, apoptosis, development, tumorigenesis, and host-pathogen interactions [19][20][21][22][23][24]. Although experiments have been performed to identify the roles of an increasing number of miRNAs, few studies have been conducted to examine the role of miRNAs in the bovine mammary gland. Here, we present a study on the role played by miR-486 in the bovine mammary gland. This study shows that the tumor suppressor PTEN is negatively regulated by miR-486 via two specific sites (nt 725-732 and nt 3,183-3,190) within the 3`UTR. MiR-486 has been sparsely studied to date, and its relevance to the mammary gland has only begun to be elucidated. Using a small RNA sequencing approach, we observed that the levels of miR-486 were higher in lactation gland tissue during the high-quality milk stage than in the low-quality BMECs after treatment with miR-486 inhibitors or negative control (Anti-NC). E: Western blotting analysis of PTEN protein expression in BMECs after treatment with miR-486 mimics or negative control (miR-NC). F: Western blotting analysis of PTEN protein expression in BMECs after treatment with miR-486 inhibitors or negative control (Anti-NC). All of the above experiments were performed in triplicate. G: Confocal microscopy analysis of PTEN expression in BMECs after treatment with miR-486 mimics (30 μM), negative control (miR-NC) ( milk and pregnancy stages and that the expression of miR-486 was almost unchanged from the low-quality lactation stage to the pregnancy stage. We further validated this result using qRT-PCR and small RNA sequencing. In situ hybridization showed that miR-486 was found primarily in mammary gland tissue and not in adipose tissue. Therefore, we concluded that the post-transfection effects of miR-486 mimics or a miR-486 inhibitor affected mammary epithelial cells more than fat cells. The results were similar for almost all stages of bovine mammary gland development. Therefore, we believe that miR-486 is a key biomarker in bovine mammary gland epithelial cells. Heterozygous PTEN mice were crossed with MMTV-wnt1 transgenic mice, resulting in the formation of breast tumors earlier in life than in the parental strains [25]. Some studies have examined the role of PTEN in normal mammary glands, but few studies have examined the secretion of these glands, which might prove that the tumor suppressor protein PTEN controls mammary gland development [17]. PTEN is commonly inactivated in human cancers and acts as a key regulator of the PIP3/AKT/mTOR pathway. One of study showed PTEN functions as an inhibitor during mammary gland development and lactation in dairy cows [26]. Indeed, our study confirmed that levels of AKT, phosphorylated AKT, mTOR and phosphorylated mTOR change in response to PTEN and that PTEN levels appeared to depend on miR-486 levels. The overexpression of miR-486 inhibited the expression of PTEN both at the mRNA and protein levels and thus increased the levels of AKT, phosphorylated AKT, mTOR and phosphorylated mTOR. PTEN is a well-known tumor suppressor in numerous cancers and normal tissues and regulates the AKT and mTOR signaling pathways. Our study provided information about the levels of nuclear PTEN. Bovine mammary epithelial cells overexpressing miR-486 had decreased PTEN expression in their nuclei; however, BMECs lacking miR-486 had increased PTEN levels. In general, despite its key role at the plasma membrane, PTEN is present in the nucleus of diverse cell types [27][28] (including tissue cells and cell lines [29][30][31]) and participates in many cellular processes that are relevant to tumorigenesis [32]. The regulation of PTEN nuclear MiR-486 Regulates Lactation and Targets PTEN Gene in Cow Mammary Gland localization is complex because PTEN plays many roles within the nucleus [17]. MiR-486 inhibited the expression of PTEN, and as same time cytoplasmic PTEN cellular staining towards nuclear staining, for PTEN/AKT/MTOR pathway plays prominent roles in development and lactation of mammary gland [26]. Here we suppose that miR-486 regulates expression of cytoplasmic PTEN, PTEN shift from cellular towards nuclear in mammary epithelial cells increase AKT/ MTOR pathway, and regulate mammary epithelial cells secretion of β-casein, triglyceride, and lactose, and plays a critical role in lactation related signaling pathways.
During the lactation period, the quality of milk that is secreted depends on milk fat (mostly triglyceride), milk protein (mostly β-casein) [33] and lactose. The results of this study show that triglyceride, β-casein and lactose levels were regulated by the transfection of miR-486 mimics or miR-486 inhibitors, indicating that miR-486 stimulated the lactation of milk fat, milk protein and lactose in the cow mammary gland.
The number and activity of mammary gland secreting cells in cows have been reported to affect the quality of milk [34]. We found that overexpressing miR-486 increased the amount of mammary epithelial cell proliferation as determined by CCK-8 assays. However, the inhibition of miR-486 showed the opposite effect. Edu incorporation experiments illustrated that miR-486 stimulated DNA proliferation by increasing the percentage of Edu-positive cells. Furthermore,  Analysis of secretion in mammary supernatants. A: Secretion of triglyceride following overexpressive miR-486, compared with negative control (miR-NC); B: Secretion of triglyceride following inhibiting miR-486, compared with negative control (Anti-NC); C: Secretion of β-casein following overexpressive miR-486, compared with negative control (miR-NC); D: Secretion of β-casein following inhibiting miR-486, compared with negative control (Anti-NC); E: Secretion of lactose following overexpressive miR-486, compared with negative control (miR-NC); F: Secretion of lactose following inhibiting miR-486, compared with negative control (Anti-NC). Data were averages of at least three independent runs; Values are means±SD, **P<0.01, ***P<0.0001. our studies showed that miR-486 regulated the cell cycle of BMECs. MiR-486 mimics increased the population of BMECs in the G0/G1 phase and decreased the population of BMECs in the S stage. Therefore, we propose that miR-486 is a novel regulator that stimulates the proliferation of BMECs. The role of miR-486 in the cell cycle is an ongoing area of study.

Conclusion
MiR-486 plays a key role in the mammary gland and directly downregulates the PTEN gene and affects downstream genes, such as AKT, mTOR, and β-casein, which play important roles in the mammary gland. Our data suggest that the repression of PTEN by miR-486 is associated with lactation and in this way increases AKT and mTOR. Additionally, miR-486 stimulates cell proliferation and increases some crucial secretory elements, such as β-casein, lactose, and lipids. Taken together, our findings indicate that miR-486 functions primarily in bovine mammary gland epithelium tissue and cells and promotes milk synthesis and secretion.