MicroRNAs (miRNAs) are endogenous, small (~22 nucleotides), and single-stranded noncoding RNAs. The role of different miRNAs in biological systems is well established. They are generally regarded as negative regulators of gene expression, as they bind to the 3′ untranslated region (3′UTR) of messengerRNAs (mRNAs), leading to mRNA degradation and/or suppression of mRNA translation [1
]. Currently, thousands of miRNAs have been identified as participating in a number of biological processes, such as cellular growth, proliferation, development, and metabolism [4
Based on Solexa sequencing, the expression of microRNA-25 (miR-25) was higher in the longissimus dorsi muscle of Large White pigs (a lean type) than in those of Tongcheng pigs (a Chinese indigenous fatty pig). Because skeletal muscle plays a vital role in whole-body metabolism [5
], we speculated that miR-25 could play a regulatory role in metabolism.
Previous studies have reported that miR-25 plays an important role in many biological processes. The expression of miR-25-3p was significantly increased in the plasma of thyroid papillary carcinoma, as compared with patients with benign tumors or healthy individuals [6
]. miR-25 expression was higher in ovarian epithelial tissue, gastric cancer, lung adenocarcinoma, and many other tumors, and miR-25 expression levels were also closely related to tumor stage and lymph node metastasis [7
]. Inhibition of miR-25 markedly improved cardiac contractility in the failing heart [11
]. miR-25 could protect cardiomyocytes against oxidative damage by downregulating the mitochondrial calcium uniporter (MCU) [12
]. Variations in miR-25 expression influenced the severity of diabetic kidney disease [13
]. However, to our knowledge, the role of miR-25 in metabolism has not been reported, and its transcriptional regulatory mechanism is not clear.
Thus, in this study, we first investigated whether miR-25 was involved in metabolism by gain-of-function and loss-of-function assays. Then, the target gene of miR-25, AKT serine/threonine kinase 1 (Akt1), which is related to metabolism, was predicted and verified using bioinformatics software and experiments. Finally, the core promoter of miR-25 was identified, and the binding of the transcription factor activator protein-2α (AP-2α) to the core promoter was shown to promote the transcriptional activity of miR-25 and downregulate Akt1 expression.
Increasing evidence shows that miR-25, a member of the miR-106b-25 cluster, is involved in many biological processes. For instance, miR-25 inhibits human gastric adenocarcinoma cell apoptosis [15
], promotes glioblastoma cell proliferation and invasion [16
], and regulates human ovarian cancer apoptosis [17
]. The miR-106b-25 cluster regulates adult neural stem/progenitor cell proliferation, migration, and differentiation [18
]. miR-25 plays an important role in heart disease [11
] and diabetic kidney disease [13
]. In addition, numerous studies have demonstrated that miRNAs are implicated in metabolism [20
]. However, miR-25 has not been functionally related to metabolism until now.
In this study, miR-25 was identified as a novel regulator of metabolism. The gain-of-function and loss-of-function assays showed that miR-25-3p inhibited the expression of PI3K
and reduced levels of triglyceride (TG), while levels of ATP and ROS were increased. PI3K has been implicated in insulin-regulated glucose metabolism [24
], and PI3K signaling has a role in many cellular processes, such as metabolic control, immunity, and cardiovascular homeostasis [25
]. It is well-known that triglycerides (TG) are a component of lipids, and participate in lipid metabolism. ATP is the most direct source of energy in an organism, and takes part in many metabolic processes. ROS, a class of single electron radicals of oxygen, comprise superoxide anions (O2−
), hydrogen peroxide (H2
), and hydroxyl radicals (·
], and are closely related to adipogenesis and myogenesis [28
]. These data indicate that miR-25-3p indeed participates in metabolism in mice.
To further understand the molecular mechanism by which miR-25-3p regulates metabolism, we searched for potential target genes of miR-25-3p via TargetScan. Fortunately, the 3′UTR of Akt1
contained a 7 nucleotides perfect match site complementary to the miR-25-3p seed region (Figure 3
B). The serine-threonine kinase ATK, also known as protein kinase B (PKB), is an important effector for PI3K signaling as initiated by numerous growth factors and hormones [32
can control glucose uptake by regulating GLUT4 in cells, thereby reducing blood sugar and promoting glycogen synthesis [32
usually promotes glycogen synthase kinase-3 alpha (GSK3α) phosphorylation and inhibits its activity [35
], and then activates glycogen synthesis [36
]. A previous study has demonstrated that overexpression of miR-25-3p downregulates Akt
expression and inactivates Akt phosphorylation in the tongue squamous cell carcinoma cell line Tca8113 [37
]. Consequently, we deduced that the role of miR-25-3p in metabolism may arise from its inhibition of Akt1
. First, the dual luciferase reporter assay demonstrated that Akt1
was a direct target of miR-25-3p, shown by the steady decrease luciferase activity of the pmirGLO-Akt1-wt vector; but not the mutant form (Figure 3
C). Meanwhile, qRT-PCR and Western blotting results showed that the expression of Akt1
was inhibited by the miR-25-3p mimics, and that this inhibition was reversed by the miR-25-3p inhibitors (Figure 3
D,E). These results suggested that the effect of miR-25-3p in metabolism was due, at least in part, to the suppression of Akt1
An increasing number of studies have shown that transcription factors are capable of binding to miRNA promoter elements and modulating miRNA transcription [38
]. Therefore, we analyzed the transcriptional mechanism of miR-25-3p in this study. Nine fragments of 5′-flanking sequences of mouse miR-25-3p were isolated. Subsequently, a series of experiments, including dual luciferase, site-directed mutagenesis, and ChIP assays, confirmed that AP-2α bound to the miR-25-3p promoter region and promoted its transcription activity (Figure 4
). Moreover, qRT-PCR and Western blotting results showed that overexpression of AP-2α resulted in the upregulation of miR-25-3p and downregulation of Akt1
, and that the knockdown of AP-2α reversed these results (Figure 5
The AP-2 family of transcription factors consists of five members, in humans and mice: AP-2α, AP-2β, AP-2γ, AP-2δ, and AP-2ε; which play important roles in several cellular processes, such as apoptosis, migration, and differentiation [41
]. AP-2α was first identified by its ability to bind to the enhancer regions of SV40 and human metallothionein IIA [43
]. Subsequently, numerous studies have demonstrated that AP-2α can regulate gene expression. For instance, AP-2α binding to the C/EBPα
promoter results in decreased C/EBPα
], and AP-2α can bind to the TACE
promoter and decrease its expression in dendritic cells [45
]. Furthermore, Qiao et al. [46
] reported that there was an AP-2α binding site in the DEK
core promoter, and overexpression of AP-2α upregulated DEK
expression. In this study, we identified that AP-2α binds to the miR-25-3p promoter region and promotes its transcription activity.
In conclusion, our results demonstrate that miR-25-3p acts as a positive regulator of the metabolism of growing C2C12 cells, by affecting Akt1
gene expression through directly binding to its 3′UTR. Moreover, the transcription factor AP-2α is able to bind to the core promoter of mouse miR-25-3p, activating mature miR-25 expression and downregulating the expression of Akt1
4. Materials and Methods
4.1. miRNA, Small RNA Oligonucleotide Synthesis, and Plasmid Construction
The miR-25-3p oligonucleotides (miR-25-3p mimics, NC, miR-25-3p inhibitors, and inhibitor-NC) and double-stranded short interfering RNAs (siRNAs) targeting AP-2α were designed and synthesized by RiboBio (Guangzhou, China).The oligonucleotides are listed in Table S1
To construct the AP-2α overexpression vector pc-AP-2α, the AP-2α coding sequence (1314 bp) was amplified from mouse C2C12 cells cDNA using the following primers: forward: 5′-CCCAAGCTTGCCACCATGCTTTGGAAACTGACGGA-3′; reverse: 5′-CCGCTCGAGTCACTTTCTGTGTTTCTCTT-3′. The PCR product was subcloned into the HindIII/XhoI sites of the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA).
The potential target site of miR-25-3p, localized in the 3′UTR of Akt1
mRNA, was predicted by TargetScan (Available online: http://www.targetscan.org/
]. The Akt1
3′UTR was amplified from C2C12 cell cDNA and inserted into the Pme
I sites of the pmirGLO vector (Promega, Madison, WI, USA). Point mutations in the seed region of the predicted miR-25-3p sites within the 3′UTR of Akt1
were generated using overlap-extension PCR [48
]. The corresponding primers are listed in Table S2
4.2. Cell Culture and Luciferase Reporter Assays
C2C12 (mouse muscle myoblast) and BHK (baby hamster kidney) cells were cultured in DMEM (Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS) (Gibco) at 5% CO2 and 37 °C.
For luciferase reporter assays, growing C2C12 or BHK cells were seeded in 48-well plates. After 12–16 h, the plated cells were transfected with a recombinant plasmid using Lipofectamine 2000 (Invitrogen). To verify the miR-25-3p targeting Akt1 3′UTR, 1 μL miR-25-3p mimics/NC was cotransfected with 0.1 μg Akt1 3′UTR/mutant plasmid into C2C12 cells. For the miR-25-3p promoter luciferase reporter assay, 0.4 μg pGL3-Basic or recombinant plasmids and 20 ng pRL-TK vector were transfected. For cotransfection luciferase assays, each well contained 0.2 μg pGL3-(Basic, miR-25-3p-P9 and AP-2α-mut), 20 ng pRL-TK, and 0.2 μg pc-AP-2α. Empty pcDNA-3.1(+) cotransfected with pGL3-(Basic, miR-25-3p-P9 and AP-2α-mut) was used as the control. After 24 h of incubation, luciferase activity was measured using a PerkinElmer 2030 Multilabel Reader (PerkinElmer, Norwalk, CT, USA).
4.3. Triglyceride Content, ATP, and Reactive Oxygen Species (ROS) Assays
For detecting the concentrations of triglyceride (TG), ATP, and ROS, growing C2C12 cells were seeded in 24-well plates the day before transfection. miR-25-3p mimic, NC, miR-25-3p inhibitor, and inhibitor-NC were transfected into confluent (~80%) cells, respectively, at a concentration of 12 nM with Lipofectamine 2000 (Invitrogen). After 24–48 h, the concentrations of TG and ATP in the lysates of cells were measured with commercial kits (Applygen (Beijing, China) and Beyotime (Shanghai, China), respectively) following the manufacturer’s instructions, and normalized to the protein content (μmol/mg protein) using the BCA assay kit (Thermo Scientific, Waltham, MA, USA). ROS were measured using the reactive oxygen species assay kit (Beyotime) following the manufacturer’s protocol.
4.4. Chromatin Immunoprecipitation (ChIP)
ChIP assays were performed to assess the binding of endogenous AP-2α to the miR-25-3p promoter in C2C12 cells using the EZ-ChIP™ Kit (Millipore, Boston, MA, USA), following a previously described method [49
]. Precleared chromatin was incubated with the AP-2α antibody (Santa Cruz Biotechnology, Dallas, TX, USA) or normal mouse IgG (Millipore) antibodies (control) overnight at 4 °C. Purified DNA from the samples and the input controls were analyzed for the presence of miR-25-3p promoter sequences containing putative AP-2α response elements using qPCR. The primers used here are listed in Table S4
4.5. RNA Isolation and qRT-PCR
For quantifying the mRNA expression of genes, growing C2C12 cells were seeded in 6-well plates. miR-25-3p mimic, NC, miR-25-3p inhibitor, inhibitor-NC, si-AP-2α, and NC were transfected into confluent (~80%) cells, respectively, at a concentration of 50 nM with Lipofectamine 2000 (Invitrogen). After 48 h, total RNA was isolated using a HP Total RNA Kit (Omega, Norcross, GA, USA) according to the manufacturer’s protocol. The cDNA was synthesized using a PrimeScript™RT reagent Kit with gDNA Eraser (Takara, Osaka, Japan) according to the manufacturer’s protocol. The qRT-PCR was performed in triplicate with iQSYBR green Supermix (Bio-Rad, Hercules, CA, USA) in a LightCycler 480 Realtime PCR machine (Roche, Basel, Switzerland). The mRNA levels of target genes were reported relative to those of the house keeping gene β-actin by using the 2−ΔΔCt
method. The qRT-PCR primers are listed in Table S5
4.6. Protein Isolation and Western Blotting
For detecting the protein expression of PI3K and Akt1, growing C2C12 cells were seeded in6-well plates. miR-25-3p mimic, NC, miR-25-3p inhibitor, inhibitor-NC, si-AP-2α, and NC were transfected into confluent (~80%) cells, respectively, at a concentration of 50 nM with Lipofectamine 2000 (Invitrogen). After 48 h, total protein was isolated using RIPA Lysis Buffer (Beyotime). The cells were washed briefly with cold phosphate-buffered saline (PBS), 150 μL RIPA Lysis Buffer (containing 1 mM PMSF) was added, incubated for 1 min at room temperature, and then centrifuged at 12,000× g for 5 min. The supernatant extract was used for Western blot analysis.
Western blot analysis was performed to analyze the expression levels of Akt1 (Affinity Biosciences, Cincinnati, OH, USA) andPI3K (Abclonal, Wuhan, China) according to the methods of Huang et al. [47
]. β-actin (Santa Cruz Biotechnology) served as the loading control.
4.7. Statistical Analysis
All the results are presented as the means ± SD. Student’s t-test was used for statistical comparisons. A p value of < 0.05 was considered to be statistically significant. ** p < 0.01; * p < 0.05; NS, not significant.