miR-25 modulates triacylglycerol and lipid accumulation in goat mammary epithelial cells by repressing PGC-1beta

Background The goat (Caprahircus) is one of the most important livestock animals. Goat milk fat is an important component in the nutritional quality of goat milk. Growing evidence points to the critical roles of microRNAs (miRNAs) in lipid metabolism. Results Using a highly sensitive method of S-poly(T) plus for miRNAs detection, we analyze the expression patterns of 715 miRNAs in goat mammary gland tissues at different stages of lactation. We observed that miR-25 expression had an inverse relationship with milk production. Overexpression of miR-25 significantly repressed triacylglycerol synthesis and lipid droplet accumulation. To explore the regulatory mechanism of miR-25 in milk lipid metabolism, we analyzed its putative target genes with bioinformatics analysis followed by 3′-UTR assays. Peroxisome proliferative activated receptor gamma coactivator 1 beta (PGC-1beta), a key regulator of lipogenics was identified as a direct target of miR-25 with three specific sites within its 3′-UTR. In addition, miR-25 mimics in goat mammary epithelial cells reduced the expressions of genes involved in lipid metabolism. Conclusions Taken together, our results show miR-25 is potentially involved in lipid metabolism and we reveal the function of the miR-25/PGC-1beta regulatory axis during lactation. Electronic supplementary material The online version of this article (10.1186/s40104-018-0262-0) contains supplementary material, which is available to authorized users.


Background
The goat (Caprahircus) is an important provider of meat and dairy products. Goat milk contains larger amounts of capric, caprylic and medium-chain fatty-acids and smaller globules [1]. These increase the digestibility of goat milk and may promote positive health effects [2]. Analysis of the human consumption of goat and cow milk fat showed that goat milk reduced cholesterol levels but not levels of triglycerides, high-density lipoprotein cholesterol, glutamic oxaloacetic transaminase or glutamic pyruvic transaminase [3]. Thus, goat milk has a higher nutritional value than cow or sheep milk.
Milk fat is a critical component in the nutritional quality of dairy products. The molecular events associated with regulation of milk fat synthesis. For example, lipogenic genes including Acetyl-CoA carboxylase 1 (ACACA), Fatty acid synthase (FASN), stearoyl-CoA desaturase (SCD), Fatty acid desaturase 1 (FADS1), FADS2, 1-acylglycerol-3phosphate O-acyltransferase 6 (AGPAT6) and glycerol-3phosphate acyltransferase, mitochondrial (GPAM) are increased until peak-lactation and decrease thereafter [4]. A deeper knowledge of lipid metabolism in the goat mammary gland during lactation is necessary to understand the features of milk, particularly the genes involved in fat metabolism.
MicroRNAs (miRNAs) are non-coding small RNAs that can post-transcriptionally regulate gene expression by pairing with the 3′-untranslated regions (3′-UTRs) or the coding regions of their target mRNAs. The base pairing between miRNA and target gene leads to either degradation of the mRNA or repression of protein translation [5]. Recently, miR-15a, miR-30e and miR-148a have been reported to regulate triacylglycerol synthesis in goat mammary epithelial cells (GMECs) by targeting low-density lipoprotein receptor-related protein 6 (LRP6), yes-associated protein 1 (YAP1) and peroxisome proliferative activated receptor gamma coactivator 1 alpha (PGC-1alpha) [6,7].
In the present study, we analyzed the miRNA expression patterns of 715 miRNAs using a highly sensitive method of S-poly(T) Plus miRNA real-time PCR [8,9]. We found that miR-25 is implicated in lipid metabolism during lactation, by directly targeting peroxisome proliferative activated receptor gamma coactivator 1 beta (PGC-1beta), which modulates the expression of sterol regulatory element-binding proteins (SREBPs). Our results establish a miR-25/PGC-1beta regulatory axis in lipid metabolism during lactation.

Animal tissue samples
Three-year-old Xinong Saanen dairy goats from Northwest A&F University experimental farm were selected and sacrificed for mammary gland tissue collection. All selected goats were of similar body weight and in non-lactation, early lactation (15 d after parturition), peak lactation (60 d after parturition) or late lactation (120 d after parturition) periods. Mammary gland tissues were immediately snap-frozen in liquid nitrogen after washing in diethylpyrocarbonate (DEPC)-treated water. All experimental procedures involving dairy goats were approved by the Institutional Animal Care and Use Committee of the College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.

RNA extraction
Total RNA of tissues and cells was extracted with RNAiso Reagent (TaKaRa, Dalian, China) according to the manufacturer's instructions. The quality of total RNA was checked by 1% agarose gel electrophoresis. The RNA was quantified using a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and stored at − 80°C before use.

Real-time PCR
Mature miRNA expression level was determined using the S-Poly (T) plus method (Geneups, Shenzhen, Guangdong, China).
For miRNA, amplification conditions were as follows: a 10-μL reaction containing 0.2 μg total RNA, 2.5 μL 4× reaction buffer, 1 μL poly A/RT enzyme mix [with 0.8 units of Poly(A) polymerase and 100 units of M-MLV High Performance Reverse Transcriptase] and 1 μL 0.5 μmol/L RT primer. The reaction was performed at 37°C for 30 min, followed by 42°C for 30 min, then 75°C for 5 min. The RT products were amplified and detected using a universal Taqman probe in a 20-μL PCR reaction containing 0.5 μL RT products, 4 μL 5× qPCR probe Mix, 0.5 units Hot Start Polymerase (FAPON, Shenzhen, Guangdong, China), 0.2 mmol/L universal Taqman probe, 0.5 μmol/L forward primer and 0.5 μmol/L universal reverse primer. Primers used were shown in Additional file 1: Table S1. 18S rRNA was used as an internal control. The primers used were as follows: 18S rRNA F: CAGCACATCTTGCGAGTACTC and 18S rRNA R: GTGCAGGGTCCGAGGTCAGAGCCACCTG GGCAATGCAGTGATGGCAAAGG.
For mRNA evaluation, 0.5 μg total RNA was synthesized into cDNA using M-MLV Reverse Transcriptase (TaKaRa) with oligo(dT) 18 plus random hexamer primers (Promega, Madison, Wisconsin, USA). Realtime PCR assays were performed with gene specific primers and SYBR Green PCR Master Mix (Applied Biosystems, Foster, CA, USA). The expression was normalized to ubiquitously expressed transcript ubiquitously expressed transcript protein (UXT).
The PCR reaction was performed at 95°C for 3 min, followed by 40 amplification cycles consisting of 95°C for 10 s and 60°C for 30 s. All real-time PCRs were performed on an ABI StepOneplus realtime PCR System (Applied Biosystems). Primers used for real-time PCR are listed in Additional file 2: Table S2. Relative expression was calculated using the 2 -△△Ct method.

Oil red O staining
Cells were washed three times with phosphate buffered saline (PBS) and then fixed in 10% paraformaldehyde for 1 h at 4°C. After two washes with PBS, the cells were stained with Oil Red O (0.5 g Oil Red O in 100 mL 70% ethyl alcohol and filtered through a 0.2 μm filter) for 1 h. Cells were then washed thrice with PBS and photographed under a light microscope.
Subsequently, 400 μL of isopropyl alcohol was added to each well, and plates were oscillatedrapidly for about 5 min. Absorbance was then measured at 510 nm. The relative fat droplet content was normalized to the control, and the results of at least three independent experiments were combined.

Triglyceride assay
The amount of intracellular triglyceride relative to total protein was detected using a tissue/cell triacylglycerol assay kit (Applygen Technologies, Beijing, China) and a BCA Protein Assay kit (Thermo Fisher Scientific, Wilmington, DE, USA), respectively.
Cells were harvested 48 h after transfection and assayed for Renilla and firefly luciferase activity using the Dual Luciferase Reporter Assay System (Promega) with a luminometer Lumat3 LB9508 (Berthold Technologies, Bad Wildbad, Germany). Firefly luciferase activity was normalized to Renilla luciferase activity.

Statistical analysis
All results are expressed as the mean ± SD (standard deviation) of at least three triplicates for each treatment. Pairwise comparisons were performed with Student's t-test using GraphPad Prism 5 software. A P-value of < 0.05 was considered statistically significant.

Differential temporal expression of miRNAs during lactation
To explore potential miRNAs involved in the regulation of lactation, we analyzed the expression patterns of 715 miRNAs from goat mammary glands at four different stages, including non-lactation, early, peak and late lactation using the S-poly(T) plus miRNA quantitative real-time PCR method (Fig. 1a). Among these 715 miRNAs, 122, 143 and 450 are from the domestic goat, sheep and cow databases, respectively (Additional file 3: Table S3). We found that 107 miR-NAs were differentially expressed between early and peak lactation (Fig. 1b), and 144 were differentially expressed between peak and late lactation (Fig. 1c). A total of 20 common miRNAs are in the 200 most abundant miRNAs and in the 100 most variable miR-NAs across lactation stages (Fig. 1d). Of these 20 miRNAs, levels of miR-17-5p, miR-25, miR-361 and miR-2340 were decreased until peak-lactation and increased thereafter (Fig. 2). Chen et al. revealed that miR-17-5p regulated lipid metabolism during goat lactation [6]. In this study, we focused on miR-25, whose expression level was the highest among these four miRNAs (Additional file 4: Figure S1).

miR-25 impaired triglyceride and lipid droplet accumulation in GMECs
Triacylglycerol droplets and are stored in the cytoplasm as micro lipid droplets [13]. To access the function of miR-25 in lipid metabolism, we analyzed the regulation of triglyceride synthesis and of lipid drop accumulation by miR-25 in goat mammary epithelial cells. We transfected epithelial cells with miR-25 mimics or mimic control (miR-NC). The efficiency of miRNA mimic transfections was confirmed by real-time PCR (Fig. 3a). We found that, miR-25 significantly reduced (0.72-fold, P = 0.0064) the synthesis of triglyceride relative to control (Fig. 3b). Moreover, Oil Red O staining assays showed a reduced number of lipid droplets in miR-25 transfected cells compared to the control (Fig. 3c). Further quantification of lipid droplets confirmed that miR-25 significantly reduced lipid droplet accumulation (0.85fold, P = 0.0213, Fig. 3d). Taken together, our results show that miR-25 has a repressive role in milk lipid metabolism.

miR-25 repressed the expression of PGC-1beta
We next explored the mechanism through which miR-25 regulates lipid metabolism by searching for mRNA targets that might mediate its effects in mammary epithelial cells. TargetScan 7.1 (Cambridge, MA, USA) predicted 1,038 genes as its target genes. Further Gene Ontology enrichment analysis using DAVID 6.8 (https://david.ncifcrf.gov) showed that 30 of these genes are lipid metabolism-associated genes  Table S4). Peroxisome proliferative activated receptor gamma coactivator 1 beta (PGC-1beta) is a well-known regulator of lipogenic genes and is required for the full transcriptional activity of SREBP [13]. To investigate whether PGC-1beta is a potential target gene of miR-25, 48 h after transfection of cells with miR-25 mimic or its control, we analyzed the mRNA and protein levels of PGC-1beta. Due to gradual increases in RNA concentration during lactation, we used UXT as a reference, which is expressed stably across time [14]. We found that miR-25 overexpression resulted in a reduction in the level of PGC-1beta mRNA (0.90-fold, P = 0.0284, Fig. 4a), while western blotting revealed posttranscriptional suppression of PGC-1beta by miR-25 (0.8-fold, P = 0.0155, Fig. 4b). These data demonstrate that PGC-1beta is a probable downstream target of miR-25.

miR-25 regulates the PGC-1beta gene by directly targeting its 3′-UTR
To understand how miR-25 affects the expression of PGC-1beta, we determined whether there is a direct interaction between miR-25 and PGC-1beta. There are three predicted miR-25 binding sites at the 3′-UTR of PGC-1beta mRNA (sites a, b and c), which are well conserved between goat, sheep, cattle and bison (Fig. 5a). As shown in Fig. 5a, we constructed two wild-type PGC-1beta 3′-UTR luciferase reporter plasmids (WT 1 and 2), and generated their corresponding mutant constructs (Mut a, b, ab and c) with seven mutated residues in the predicted binding sites by site-directed mutagenesis. Enhanced expression of miR-25 significantly repressed the luciferase activities of WT1 and WT2 reporters (0.77-fold, P = 0.0012 and 0.78-fold, P = 0.0017, respectively), whereas these repressions were partly abrogated by Mut a, b or c (Fig. 5b). Moreover, the repression of WT1 was completely abolished when both site a and b were mutated (Fig.  5c). Based on these results, we conclude that miR-25 repressed PGC-1beta expression in goat mammary epithelial cells by directly targeting its 3′-UTR.

Conclusion
In conclusion, we revealed miRNA expression patterns in goat mammary gland tissue during lactation and identified miR-25 as lactation related miRNA. We then characterized the role of miR-25 in triglyceride and lipid droplet accumulation and lipid metabolism-related gene expression in GMECs, and determined that miR-25 can repress lipid synthesis via PGC-1beta in GMECs during lactation.