Human HCC tissues and adjacent para-carcinoma tissues were collected from fifty-seven HCC patients undergoing surgical operations at the Xiangya Hospital of Central South University. The clinical research was approved by the Ethics Committee of the Xiangya Hospital of Central South University. Informed consent forms were approved by all of the patients.
Cell culture and drugs.
Human HCC cell lines (MHCC97H, HepG2, Huh7 and SMMC7721) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in DMEM with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified incubator (Thermo, USA), 5% CO2, 95% air atmosphere. ATL was purchased from Sigma-Aldrich (cat. no. A2987; Merck Millipore, Darmstadt, Germany). The molecular structure of Atractylenolide III was shown in Figure 1A.
Cell transfection and plasmid constructs.
Small interfering RNA (siRNA) was constructed to specifically silence FGFR1. siRNAs were synthesized by GenePharma (Shanghai, China). Target site in human gene encoding FGFR1 was as following: siRNA-FGFR1 sense strand, 5′-GCTTCTTTCCAGCCTCTTT-3′, the siRNA-FGFR1 antisense strand, 5′-AAAGAGGCTGGAAAGAAGC-3′; siRNA-NC sense strand, 5′-AACTCCGGTCGAGGAGGAC-3′, the siRNA-NC antisense strand, 5′-GTCCTCCCTCGACCGGAGTT-3′. siRNA-FGFR1 and siRNA-NC were transfected into HepG2 and SMMC7721 cells using Lipofectamine™ 2000 (Invitrogen, CA, USA). miR-195-5p mimics (5’-UAGCAGCACAGAAAUAUUGGC-3’) and scramble sequence (5’-UCAUGUAGGUAAGUGCGACGA-3’) were synthesized by RiboBio (Guangzhou, China) and transfected with into HepG2 and SMMC7721 cells with a final concentration of 100 nM using Lipofectamine 2000 (Invitrogen) for 48 h at 37 ℃ according to the manufacturer’s protocol. human FGFR1 plasmids without 3'-UTR, which did not contain the conserved complementary sequence binding with miR-195-5p, were purchased from GeneCopoeia, Inc. (Rockville, MD, USA). An empty plasmid served as negative control (vector-Con). FGFR1 overexpressed plasmids and vector-Con were transfected using Lipofectamine 2000 for 48 h at 37˚C, according to the manufacturer’s protocols.
Luciferase reporter assay.
FGFR1 wild-type (WT) or mutant-type (Mut) 3'-UTR was inserted into the multiple cloning sites of the luciferase-expressing pMIR-REPORT vector (Ambion). For the luciferase assay, HepG2 and SMMC7721 cells containing the WT or Mut 3'-UTR of FGFR1 (0.5 μg) were co-transfected with miR-Con or miR-195-5p mimics using Lipofectamine 2000. The luciferase activity was measured using a luciferase reporter assay kit (Promega Corporation, Madison, WI, USA).
Cell viability detection by CCK-8.
The CCK-8 assay (Dojindo Japan) was performed as previously described , human HCC cells (1×104) were seeded in the 96-well plate and incubated with ATL (0, 10, 100 or 500 μM) for 24 hours, 48 hours and 72 hours. Absorbance was recorded at 450 nm using Elx800 Reader (Bio-Tek Instruments Inc., Winooski, VT, USA).
Migration and invasion assays.
Cells (2 x 104) migration was analyzed using the transwell chamber (8 μ pore size; Corning Incorporated, Corning, NY, USA) without the Matrigel matrix. Cells (2 x 104) were seeded into the upper chamber pre-coated with Matrigel matrix (BD Biosciences) for invasion analysis. After incubation for 24 h, cells in the down chamber were stained with 0.1% crystal violet (Beyotime) and photographed by an inverted fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).
After HepG2 and SMMC7721 cultured with different conditions, cells were collected and fixed on the glass slide. TUNEL (Beyotime Institute of Biotechnology, Haimen, China) assay was performed according to the manufacturer’s instructions. TUNEL positive cells were mounted under a fluorescence microscope (Olympus, Japan).
Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). TaqMan® RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) and TaqMan® MicroRNA assay (Applied Biosystems; Thermo Fisher Scientific, Inc.) were utilized to detect the expression of miRNAs, according to the manufacturer’s protocol. U6 small nuclear RNA was used as an endogenous control.
Primary antibodies of FGFR1 (cat. no. ab824; dilution, 1: 1000; Abcam, Cambridge, MA, USA) and β‑actin (cat. no. sc‑81178; dilution, 1: 2000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used to incubate membranes. The membranes were washed three times with TBST and incubated with secondary antibodies donkey anti‑mouse IgG (sc‑2096, dilution, 1:10,000; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature and visualized with an Amersham ECL Western blotting detection reagent (GE Healthcare Life Sciences, Chalfont, UK).
Immunohistochemical (IHC) staining
The experimental procedures of IHC staining and the scoring rules of FGFR1-positive staining in HCC tissues and adjacent para-carcinoma tissues were referred to as previously described . Primary antibody of FGFR1 (cat. no. ab824; dilution, 1: 100) was obtained from Abcam (Cambridge, UK).
Data from these experiments were reported as mean ± standard deviation for each group. All statistical analyses were performed using PRISM version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). The student t-test was used to analyze two-group differences. Inter-group differences were analyzed by one-way ANOVA. Differences with a P value of < 0.05 were considered statistically significant.