Cells and treatments
Normal human hepatocyte cell cline (L-02 cells) and three HCC cell lines (Hep3B cells, HepG2 cells, and Huh7 cells) were obtained from iCell (China) and cultured in DMEM medium containing 10% FBS, which were incubated under 37℃and 5% CO2. To obtain OPN-overexpressed cells, Hep3B cells were transfected with adenovirus containing pcDNA3.1-OPN, with pcDNA3.1-NC as a negative control. After 48 h transfection, cells were collected and the transfection efficacy was identified using the Western blotting assay.
CCK-8 assay
Cells were implanted in 96-well plates for 24 h, followed by adding with 10 µl CCK8 solution. After incubating for 2 h, the OD value was detected using the microplate reader (CMaxPlus, MD, USA).
Western blotting assay: The BCA kit (pc0020, Solarbio, China) was utilized to quantify the protein isolated from cells, followed by being separated with the 12% SDS-PAGE. The separated protein was transferred from the gel to the PVDF membrane, which was further introduced with 5% skim milk. Then, the membrane was introduced with the primary antibody against PI3K (1:1000, AF6241, Affinity, USA), p-AKT (1:1000, AF0016, Affinity, USA), AKT (1:1000, AF6261, Affinity, USA), OPN (1:2000, AF0227, Affinity, USA), Twist (1:1000, AF4009, Affinity, USA), E-cadherin (1:2000, AF0131, Affinity, USA), N-cadherin (1:1000, AF4039, Affinity, USA), and GAPDH (1:10000, AF7021, Affinity, USA). The second antibody (1:6000, 7074, CST, USA) was subsequently added to be incubated for 90 min. Finally, ECL reagent was added to expose the bands, which were further quantified with the Image J software.
Wound healing assay
When the cell density reached more than 90%, 200 µL of the gun tip was used to scratch in each well, followed by discarding the medium and replaced with DMEM incomplete medium. Then the scratch in each well was photographed. Cells were put into the incubator, and the scratch of each well was photographed again after 24 hours. According to the scratch condition, the 24 h scratch data and the 0 h scratch data were determined. The corresponding scratch width and migration rate was calculated.
Clone formation assay: 2000 cells/well were implanted in 6-well plates and incubated for 10 days. When macroscopic cloning appeared, the supernatant was aspirated and cells were fixed with the mixture of methanol and acetic acid at a ratio of 3:1 at room temperature for 5 min. Methanolic solution containing crystal violet was added and fixed for 15 min. The supernatant was aspirated and air-dried at room temperature for observation using an optical microscope (AE2000; Motic, China).
Transwell assay
The upper chamber of the Transwell insert (3422; Corning, USA) was implanted with 1.5 × 105 cells cultured in serum-free medium, which were then filled with 20% FBS-supplemented medium in the lower chamber. After 24 h incubation, cells were wiped off from the upper chamber, and those in the lower chamber were stained with crystal violet. Finally, the migrated cells were counted using an optical microscope (AE2000; Motic, China).
The detection of apoptosis using the flow cytometry
1×106 cells were collected and washed by PBS buffer, which were then re-suspended with 300 µl pre-cold 1×Annexin V-FITC binding buffer. Then, cells were introduced with 5 µl Annexin V-FITC reagent and 10 µl PI reagent, followed by 10 min incubation in the dark at room temperature. Lastly, cells were loaded onto the flow cytometry (C6, BD, USA) for the analysis of apoptosis.
Animals and xenograft model: 24 female nude mice (7–9 week) were purchased from Charles River (China). After 7 days of adaptive feeding, nude mice were randomly divided into 4 groups: Control, 100 mg/kg Fenofibrate, 200 mg/kg Fenofibrate, and OPN OE + 200 mg/kg Fenofibrate groups. 6 nude mice were used in each group. For the establishment of xenograft model, 5*106 cells were inoculated subcutaneously into the back of the axilla per mouse with a volume of 0.25mL/ mouse. The tumor volume was recorded every three days. The administration was performed until the tumor volume reached approximately 100 mm3. In the control group, the Hep3B cell xenograft model was established, followed by orally dosed with normal saline for 14 days. In the 100 mg/kg Fenofibrate and 200 mg/kg Fenofibrate groups, the Hep3B cell xenograft model was established, followed by orally dosed with 100 mg/kg Fenofibrate and 200 mg/kg Fenofibrate daily for 14 days, respectively. In the OPN OE + 200 mg/kg Fenofibrate group, the OPN-overexpressed Hep3B cell xenograft model was established, followed by orally dosed with 200 mg/kg Fenofibrate daily for 14 days. Tumors were weighed and sampled at the end.
Statistical analysis
Mean ± SD was utilized to present data, which was analyzed using the one-way ANOVA method with the software of GraphPad Prism 7.0 software. P < 0.05 was considered to be a statistically significant difference.