miR-25 modulates triacylglycerol and lipid accumulation in goat mammary epithelial cells by repressing PGC-1beta
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.
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.
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.
KeywordsGoat mammary epithelial cells Lipid miR-25 PGC-1beta Triacylglycerol
Acetyl-CoA carboxylase 1
1-acylglycerol-3-phosphate O-acyltransferase 6
Fatty acid desaturase 1
Fatty acid synthase
Goat mammary epithelial cells
Glycerol-3-phosphate acyltransferase, mitochondrial
Insulin induced gene 1
Low-density lipoprotein receptor-related protein 6
Peroxisome proliferative activated receptor gamma coactivator 1 beta
Peroxisome proliferator-activated receptor gamma
Sterol regulatory element-binding protein 1
Sterol regulatory element-binding protein 2
Sterol regulatory element-binding proteins
Ubiquitously expressed transcript protein
Yes-associated protein 1
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 . These increase the digestibility of goat milk and may promote positive health effects . 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 . 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-3-phosphate O-acyltransferase 6 (AGPAT6) and glycerol-3-phosphate acyltransferase, mitochondrial (GPAM) are increased until peak-lactation and decrease thereafter . 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 . 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.
Cell culture and transfection
Goat mammary epithelial cells (GMECs) were isolated from mammary gland tissue, and purified as previously reported . These cells were treated in a lactogenic medium for 48 h to induce differentiation into secretary cells [11, 12]. For cell experiments, GMECs were cultured in DMEM/F12 medium (Hyclone Laboratories, Beijing, China), containing 5 μg/mL insulin, 5 μg/mL hydrocortisone, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 ng/mL epidermal growth factor 1 (EGF-1, Gibco, Gaithersburg, MD, USA), and 10% fetal bovine serum (FBS, Biological Industries, BeitHaemek, Israel) in a humidified incubator with 5% CO2 at 37 °C. Synthetic miRNA mimics were purchased from RiboBio (Guangzhou, Guangdong, China) and transfected into GMECs using the K2 transfection system (Biontex Laboratories GmbH, München, Germany) according to manufacturer’s instructions.
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.
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: GTGCAGGGTCCGAGGTCAGAGCCACCTGGGCAATGCAGTGATGGCAAAGG.
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). Real-time 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 real-time 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.
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.
3′-UTR luciferase reporter assay
We applied TargetScan (http://www.targetscan.org) to predict targets and miRNA binding sites. To generate reporter constructs for luciferase assays, the 3′-UTR of PGC-1beta was PCR amplified from goat genomic DNA and inserted into the pmirGLO dual-luciferase vector (Promega). The primers used were as follows: PGC-1beta WT-1, 5′-CCAGAATTCTCTTCTCCCCATTACACCTTGACCC-3′ (forward) and 5′-CCACTCGAGTCCATTTACCCTGTACCCCTGGACT-3′ (reverse); PGC-1beta WT-2,5′-CCAGAATTCGACTGTATCCACCAGCTACCCAGAT-3′ (forward) and 5′-CCACTCGAGATTCCTCAAGAAACAAAGTTGGGAG-3′ (reverse). To construct mutated 3′-UTR reporter vectors, the predicted miRNA binding sites were mutated by site-directed mutagenesis with the following primers: Muta,5′-CTTTATGTGGGAAGAGAACGTTATAGAAATCTGTCT-3′ (forward) and 5′-TCAGCGAGACAGATTTCTATAACGTTCTCTTCCCACA-3′ (reverse); Mut b,5′-ACCTGGCTAGTGCTTATGACGTTATTGTTTAAGCTGG-3′ (forward) and 5′-TGGGGCCCAGCTTAAACAATAACGTCATAAGCACTAG-3′ (reverse); Mut c,5′-GGAGGGGTTTACTGTAACACGTTATCTGGCAGCCCAG-3′ (forward) and 5′-CAGCAGCTGGGCTGCCAGATAACGTGTTACAGTAAAC-3′ (reverse). All constructs were confirmed by sequencing.
For PGC-1beta 3′-UTR luciferase assays, co-transfection of GMECs with PGC-1beta 3′-UTR or mutated PGC-1beta 3′-UTR reporter plasmids and miR-25 mimics or mimic control was performed with the PEI transfection reagent following the manufacturer’s instructions.
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.
For western blot analysis, cells were collected and lysed with ice-cold RIPA buffer (50 mmol/L Tris-HCl, pH 7.5; 150 mmol/L NaCl; 1% NP-40; 0.25% sodium deoxycholate, 1 mmol/L EDTA), supplemented with PMSF (Sigma-Aldrich, St. Louis, MO, USA).
Protein concentration was determined using a BCA Protein Assaykit (Thermo Scientific). Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes (Millipore, USA) and probed with the primary antibodies: polyclonal rabbit anti-PGC-1beta (Cat #22378-1-AP, 1:500 dilution; Proteintech Group) or polyclonal rabbit anti-GAPDH (Cat #HS-PP-0640, 1:1,000 dilution; Hanlin Biotech, Shijiazhuang, China). Polyclonal goat anti-rabbit IgG-HRP (Bio-Rad, Hercules, CA, USA) was used as secondary antibody. The protein bands were visualized using the chemiluminescent ECL western blot detection kit (Advansta, Menlo Park, CA, USA), and images were captured with the Tanon-5200 imaging system (Tanon, Shanghai, China).
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
miR-25 impaired triglyceride and lipid droplet accumulation in GMECs
miR-25 repressed the expression of PGC-1beta
miR-25 regulates the PGC-1beta gene by directly targeting its 3′-UTR
miR-25 modulated other lipid metabolism-related genes in GMECs
In this study, we analyzed the expression of 715 miRNAs in the goat mammary gland during lactation and characterized miR-25 as a repressor in lipid-metabolism. Our results revealed that miR-25 overexpression leads to repression of PGC-1beta by direct targeting of three different regions within the PGC1-beta 3′-UTR. This reduces the expression of certain lipogenic genes, triglyceride synthesis and lipid droplet accumulation.
miR-25 belongs to the miR-25-93-106b cluster, which plays an important role in many malignancies, including ovarian cancer , cervicalcancer , cholangiocarcinoma  and lung cancer [25, 26]. There is growing evidence that miR-25 is involved in lipid metabolism. Liang et al.  showed that miR-25 is downregulated during adipocyte differentiation and suppressed 3 T3-L1 adipogenesis by targeting Kruppel-like factor 4 and CCAAT/enhancer-binding protein alpha. Moreover, Hsieh et al. found that miR-25 was downregulated in high-fat diet fed mice relative to low-fat diet fed mice . When viewed together, these findings and our results provide strong evidence to support the involvement of miR-25 in lipid metabolism.
PGC-1beta has long been shown to stimulate the expression of genes involved in lipid metabolism via direct co-activation of the SREBP family . In the present study, decreased SREBP-1c expression by miR-25 overexpression suggests that miR-25 modulates lipid metabolism, at least partially, via the PGC-1beta/SREBP pathway. On the other hand, miR-25 represses target PGC-1beta by binding to multiple sites within its 3′-UTR. Multiple binding sites for the same miRNA can enhance the degree of modulation . In addition, the three miR-25 binding sites within the PGC-1beta 3′-UTR are highly conserved among ruminants, highlighting miR-25 as regulator of PGC-1beta protein expression. These data indicate the importance of miR-25 in regulating PGC-1beta expression.
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.
We would like to thank Northwest A&F University for providing the goat mammary gland tissues and GMECs. We thank Jeremy Allen, PhD, from LiwenBianji, Edanz Group China, for editing the English text of a draft of this manuscript.
This work was supported by the Transgenic Project from the Ministry of Agriculture [2014ZX08009-051B to JL], the National Natural Science Foundation of China [81370151 and 81570046 to DG, 31701185 to HQ and 81700054 to YZ]; the Shenzhen Municipal Basic Research Program [JCYJ20150729104027220 to DG and JCYJ20160520174217859 to HQ]; Shenzhen University Interdisciplinary Innovation Team Project [000003 to DG]; Natural Science Foundation of Guangdong Province [2017A030310450 to HQ]; Research Project of Shenzhen Technology University [201731 to HQ].
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its Additional files.
HQ and DG conceived and designed the experiments. LM, ZC and HQ performed the experiments. HQ, JL, LL and DG provided expert advice. All authors analyzed the data. HQ and LM wrote the paper and LL and DG reviewed the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The use of animals was in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of China.
The authors declare that they have no competing interests.
- 10.Shi H, Luo J, Zhu J, Li J, Sun Y, Lin X, et al. PPAR gamma regulates genes involved in triacylglycerol synthesis and secretion in mammary gland epithelial cells of dairy goats. PPAR Res. 2013;310948:2013.Google Scholar
- 22.Zhang H, Zuo Z, Lu X, Wang L, Wang H, Zhu Z. miR-25 regulates apoptosis by targeting Bim in human ovarian cancer. Oncol Rep. 2012;27:594–8.Google Scholar
- 25.Xiang J, Hang JB, Che JM, Li HC. miR-25 is up-regulated in non-small cell lung cancer and promotes cell proliferation and motility by targeting FBXW7. Int J Clin Exp Pathol. 2015;8:9147–53.Google Scholar
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