Adult (age, 6 weeks) male C57BL6/J mice were obtained from the Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). The mice were fed with free access to water and food in plastic cages at a controlled temperature of 20± 2℃, humidity of 50%–60% and a 12-hour light–dark cycle. After 1 week of acclimatization, the mice were randomly divided into 3 groups: control group (n=10), HFD-fed group (n=10), and fenofibrate + HFD-fed group (n=10), fenofibrate was dissolved with 0.5% sodium carboxymethyl cellulose (CMC-Na). The mice in control group were fed with normal chow diet, and the mice in HFD-fed group were fed with a high fat diet (HFD), which consists of 20% carbohydrate, 20% protein and 60% fat (total 25.07 kJ/g), for 14 weeks. For the fenofibrate +HFD-fed group, the mice fed HFD were orally gavaged with fenofibrate (40 mg/kg) daily for the last 4 weeks. Body weight was measured once a week throughout the investigation.
Mouse liver was homogenized in liquid nitrogen, the homogenate was lysed on ice for 1 h in lysis buffer (BioTeKe, Beijing, China). Protein lysates were loaded into each well and separated on 7.5%, 10% or 12.5% SDS polyacrylamide gel. Separated proteins were then transferred to immobilon-PSQ transfer PVDF membrane (Millipore, Bedford, MA), and blocked with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween-20 (TBST) for 2 hours at room temperature. The primary antibodies were anti-ATP2A2/SERCA2b (#4388, Cell Signaling Technology, Beverly, MA), anti-Bip (#3183, Cell Signaling Technology), anti-CHOP (#2895, Cell Signaling Technology), and anti-GAPDH antibodies (#8884, Cell Signaling Technology). The immunoreactive signals were detected using the ECL western blotting substrate reagents (#32109, Thermo Scientific Science, Waltham, MA). An imaging system (Amersham Imager 600) was used for documentation of the western blotting results. Quantitation was analyzed with the ImageJ (NIH).
Total miRNAs in tissue were extracted with the miRcute miRNA Isolation kit (DP501, Transgen, Being, China). cDNA was reversely synthesized using the miRcute Plus miRNA First-Strand cDNA kit (KR211-01, Trangen, Being, China). Quantitative real-time PCR (qRT-PCR) analysis of let-7a, let-7b, let-c, let-7d, let-7e, let-7f, let-7g, let-7i, and mir-98 were performed using miRcute Plus miRNA qPCR kit (SYBR Green) (FP411-01-01,Trangen, Being, China) on a Roche LightCycler 480 System (Roche Applied Science, Mannheim, Germany). The primers used for PCR were as follows:
let-7a: 5’- UGAGGUAGUAGGUUGUAUAGUU- 3’;
let-7b: 5’- UGAGGUAGUAGGUUGUGUGGUU- 3’;
let-7c: 5’- UGAGGUAGUAGGUUGUAUGGUU - 3’;
let-7d: 5’- AGAGGUAGUAGGUUGCAUAGUU- 3’;
let-7e: 5’- UGAGGUAGGAGGUUGUAUAGUU- 3’;
let-7f: 5’- UGAGGUAGUAGAUUGUAUAGUU- 3’;
let-7g: 5’- UGAGGUAGUAGUUUGUACAGUU- 3’;
let-7i: 5’- UGAGGUAGUAGUUUGUGCUGUU- 3’;
mir-98: 5’- UGAGGUAGUAAGUUGUAUUGUU- 3’.
All qRT-PCRs were performed in triplicates. U6 was used for normalization.
Luciferase reporter assay
The 3′-UTR of SERCA2b mRNA was amplified by PCR and cloned into the psiCHECK2 luciferase reporter vector. The human hepatocarcinoma cell line HepG2 cells were grown in 24-well plate containing Dulbecco’s Modified Eagle Medium (Hyclone, Logan, UT) supplemented with 10% Fetal Bovine Serum (FBS, Hyclone), and maintained at 37°C with 5% CO2. When 60% to 80% confluent, the cells were transfected using Lipofectamine 2000® Reagent (Thermo Fisher Scientific, Waltham, MA) with 3′-UTR of SERCA2b reporter plasmids with negative control (NC) mimics or miR-let-7 mimic. At 48 h transfection, the luciferase activity was determined by the Dual Luciferase Assay System (Promega, Beijing, China). The renilla luciferase activity was used as a normalization in each well. All experiments were repeated three times.
Hematoxylin and eosin (H&E) staining
Fresh liver samples were fixed in 10% formalin and embedded in paraffin. After sectioned at a 5-μm thickness, and the sections were mounted onto glass microscope slides, and air-dried at room temperature for 24 h. The sections were then stained with hematoxylin and eosin (H&E). Images were acquired in a Leica aperio CS2 system.
Oil-red O staining
Oil-red O staining was used to determine lipid deposition. In briefly, OCT-embedded tissues were sectioned at a 10-μm thickness, and fixed in 10% formalin for 10-15 min, then rinsed in distilled water and air-dried. The sections were stained with freshly prepared Oil Red O staining solution (Sigma-Aldrich) for 8–10 min at 60℃. Finally, the sections were counterstained with hematoxylin. Images were acquired in a Leica aperio CS2 system.
The data presented in each figure are mean ± SD of three independent experiments performed in triplicate. The data presented in each figure are mean ± SD. Statistical differences between two groups were analyzed using Student’s t-test. Statistical difference between multiple groups were performed by one-way ANOVA, followed by a Student-Newman–Keuls test. Data were analyzed using GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA), A value of P < 0.05 was considered to represent a statistical significance.