Study design and participants
Ten HCC tissues and adjacent noncancerous tissues were used for the analysis of lipid compositions. HCC tissues and corresponding adjacent noncancerous tissues were used for western blotting to detect the levels of β-catenin and FXR. Patient tissues were obtained from the Affiliated Hospital of Guizhou Medical University between July 2020 and January 2021. Primary HCC was diagnosed. These patients did not receive any treatment before surgery. Patient tissues and their medical records were used in this study with their consent. Ethical permission of this study was obtained from the Ethics Committee of the Affiliated Hospital of Guizhou Medical University (NO. 2019–231).
GC/MS analysis for fatty acids
Lipid extraction for GC/MS analysis was conducted at LC Bio (Hangzhou, China). Tissues (20 mg) were grinded with 1 ml of cold methanol using TissueLyser at 50 Hz for 90 s. Then, 40 μl homogenate, 10 μl internal standard (0.2 mg/ml C17:0-d33, 0.4 mg/ml BHT) methanol solution and 200 μl methanol-hexane (4:1, v/v) were mixed and subsequently placed in liquid nitrogen for 10 min. After adding 20 μl acetyl chloride, the mixture was kept in the dark for 24 h. Then, the mixture and 0.5 ml of 6% K2CO3 solution were cultured in an ice bath to terminate the reaction and neutralize the hydrochloric acid produced in the reaction. Then, 50 μl of N-hexane was added to the mixture and centrifugated at 3000 rpm for 10 min. Finally, the top layer was collected for GC/MS analysis.
All samples were analysed on a GC/MS (7890b-5977b, GC/MS, Agilent, USA). The chromatographic conditions were as follows: chromatographic column, Agilent DB-225 (10 m × 0.1 mm ID × 0.1 µm); temperature of the injection port and detector, 230 °C; and sample injection volume, 1 μL. The mass spectrometry conditions were as follows: sampling mode, SCAN; ion source temperature, 230 °C; and quadrupole temperature, 150 °C. The data acquisition and analysis were conducted in Mass Hunter software (Version B.08.00, Agilent, USA).
HCC cell lines
HepG2, Huh7 and L02 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). DMEM (GibcoBRL, USA) medium was used to culture cell lines at 37 °C with 5% CO2.
Cell Counting Kit-8(CCK-8) assay
The CCK-8(Dojindo) assay was employed to analyse the proliferation. HCC cells (3000/well) were seeded into 96-well plates with 100 µl complete medium (control) or 100 µl different concentrations of alpha-linolenic acid (Sigma–Aldrich, St. Louis, MO, USA). HCC cells (3000/well) were seeded into 96-well plates with 100 µl complete medium (control) or 100 µl different concentrations of GW4064 (the FXR agonist, CAS No.: 278779-30-9, MCE). For the OD value testing, each well was cultured with 10 µl of CCK-8 reagent for 2 h. The OD values were measured at 450 nm with a SYNERGY H4 microplate reader.
Cells were seeded in 6-well plates containing coverslips. When grown to 60% confluency, cells were cultured with 5-ethynyl-2′-deoxyuridine (EdU-594, Beyotime, China) for 2 h. Phosphate-buffered saline washed the cells three times for 5 min and then cells were fixed with 4% formaldehyde for 10 min. Next, the cells were permeabilized with 0.3% Triton X-100 (Sigma, USA) for 15 min. Then, the cells were incubated with click reaction liquid for 30 min and subsequently incubated with Hoechst 33,342 for 10 min without light. Finally, the coverslips containing cells were photographed by a Zeiss Laser Microscope.
Cell migration and invasion assays
When cells grow to 100% confluency in 6-well plates, wounds are generated by a 200 µl pipette tip. These photos of wounds serve as the time point of 0-h. Cells were cultured with or without alpha-linolenic acid in free serum medium for 48 h, and the photos of wounds were collected. For the Transwell assay, the cells were seeded with 2 × 104 cells/well into the upper compartment without serum in Transwell chambers (8 µm pore size, Corning Incorporated). The upper compartments precoated with Matrigel are used for invasion assay, and upper compartments for the migration assay are without Matrigel. And the lower compartment contains 10% foetal bovine serum with 200 µM alpha-linolenic acid (700 µl). The cells are fixed with 4% paraformaldehyde and stained with 0.1% crystal violet after incubation for 48 h. Cells from the upper compartment were wiped away with cotton swabs. An inverted microscope is used to photograph the migrated cells.
Total proteins were extracted with RIPA buffer containing protease inhibitor, and the protein concentration was tested by a BCA kit (Solarbio, Beijing, China). Proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. 5% milk blocked the membrane for 1 h at room temperature, and then primary antibody was used to interact with target protein at 4 °C overnight. Then second antibody interacted with primary antibody for 1 h at room temperature. The primary antibodies included anti-β-catenin (1:9000, 51,067-2-AP, Proteintech, China), anti-NR1H4 (1:2000, 25,055-1-AP, Proteintech, China), anti-GAPDH (1:7000, 10,494-1-AP, Proteintech, China), anti-cyclin D1 (1:1000, 26,939-1-AP, Proteintech, China) and anti-c-myc(1:8000, 10,828-1-AP, Proteintech, China). The secondary antibody was goat anti-rabbit IgG (1:6000, SA00001-2, Proteintech, China).
Normality of distribution was evaluated by the Kolmogorov–Smirnov test. Student’s t test was performed based on the normally distributed continuous data. The data were not normally distributed, so the Mann–Whitney U test was adopted. Statistical analysis and graph production were performed with GraphPad Prism 8 (GraphPad Software, USA). A p value less than 0.05 was served as statistical difference.