Myricetin (cat. no. PS1149-0025; Chengdu Pushi Biotechnology Co., Ltd.; purity, 98%), was maintained at −20°C in an appropriate storage concentration (3.14×10⁴ µmol/l), dissolved with dimethylsulfoxide (DMSO). The working solution of the drug was obtained by adding the corresponding amount of high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fetal bovine serum (FBS) was obtained from Shanghai Luoshen Biotechnology Co., Ltd. (Shanghai, China). Radioimmunoprecipitation assay (RIPA) buffer, trypsin, MTT and SDS-PAGE (10% gels) were used (Beyotime Institute of Biotechnology, Haimen, China). Mouse anti-human GAPDH (cat. no. 51332), rabbit anti-human epithelial (E)-cadherin (cat. no. 3195), rabbit anti-human neural (N)-cadherin (cat. no. 13116), rabbit anti-human vimentin (cat. no. 5741) and the secondary antibodies, including goat anti-rabbit (cat. no. 6990) and goat anti-mouse (cat. no. 5946) conjugated to horseradish peroxidase (HRP) were used (all from Cell Signaling Technology, Inc., Danvers, MA, USA).

Human HCC MHCC97H cell lines (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) were cultured in high-glucose DMEM containing 10% FBS and maintained at 37°C in a humidified atmosphere containing 5% CO_2.

MHCC97H cells in the logarithmic growth phase were isolated, digested with 0.25% trypsin cell digestive fluid (cat. no. C0201; Beyotime Institute of Biotechnology) into a single cell suspension and seeded onto a 96-well plate (1×10⁴ cells/well), allowed to adhere overnight at 37°C in a humidified atmosphere containing 5% CO₂. Various concentrations (0, 10, 20, 30, 40, 50, 100 and 200 µmol/l) of myricetin were added into 96-well plates, which were divided into 7 groups with 6 wells for each group. Subsequent to being cultured for 48 h, 10 µl of MTT (5 mg/ml) solution was added to each well of the plate and the cells were cultured for 4 h. A total of 150 µl DMSO was added to each well. Plates were incubated for 15 min on the table at 100 rev/min in dark. The absorbance was measured with a spectrophotometer at a wavelength of 490 nm and the cell viability curve was obtained. All experiments were performed in triplicate and repeated thrice.

Hepatoma cells in the logarithmic growth phase were seeded onto a 6-well plate (1×10⁶ cells/well). When the cells reached 70–90% confluency, they were transferred to the serum-free DMEM and cultured overnight at 37°C in an atmosphere containing 5% CO₂. On the following day, a scratch wound was created in the cell monolayer with a 10-µl pipette tip, and the scratch state at the beginning of cell culture (0 h) was observed and photographed under a light microscope at ×40 magnification. The serum-free DMEM containing corresponding concentrations (0, 10, 20, 30, 40, 50, 100 and 200 µmol/l) of myricetin was subsequently added to the 6-well plates. Cells were photographed after being cultured for 24 and 48 h at 37°C under an inverted phase contrast fluorescence microscope at ×40 magnification. Five visual fields were counted for each experimental group and all the experiments were repeated thrice.

Cell migration experiments were performed with the HCC MHCC97H cell lines, cultured overnight in serum-free DMEM and isolated into a single cell suspension. The Transwell chamber was placed in a 24-well plate and 600 µl of complete medium supplemented with 10% FBS containing 0, 25, 50 and 100 µM myricetin was added to the lower chamber of the Transwell. Subsequently, 200 µl of serum-free cell suspension containing 0, 25, 50 and 100 µM myricetin was added to a total of 2.5×10⁵ cells/ml in the upper chamber of the Transwell. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h. The Transwell chamber was removed and soaked in Giemsa stain subsequent to rinsing with PBS (Jiangsu Lvye Biotechnology Co., Ltd., Shanghai, China; solution A contained 1% methylene blue, 1% eosin, 1% azure, glycerin and methanol, and was used at 25±1°C for 2 min; solution B contained 0.2 mol monometallic sodium orthophosphate and 0.2 mol disodium hydrogen phosphate, pH was 6.4±0.2, and was used at 25±1°C for 8 min). The non-migratory cells in the upper chamber of the Transwell were removed gently with a cotton bud following rinsing with PBS. The cells at the bottom of membrane were observed, photographed and counted under inverted phase contrast microscope. Six visual fields (magnification, ×100 were randomly counted for each well. Two wells were set for each group and all the experiments were performed in triplicate and repeated thrice.

The Matrigel was placed in the refrigerator overnight (8 h) for defrosting and the upper surface of membrane was covered with 100 µl Matrigel. Cells at a density of 1×10⁶ cells/well were plated in the upper chamber in serum-free DMEM. Subsequent to incubation for 8 h at 37°C in an atmosphere containing 5% CO₂, invaded cells were stained with Giemsa stain for 10 min at 25°C and counted under an inverted phase contrast microscope (×100 magnification) Two wells were set for each group and of all the experiments were performed in triplicate and repeated thrice.

MHCC97H cells were seeded in a 6-well plate and cultured at 37°C in a humidified atmosphere containing 5% CO₂. The complete medium containing 0, 25, 50 and 100 µM myricetin was added into the well. The cells were cultured for 48 h, when the cell reached 70% confluency. The cover slip was removed and the plate was rinsed twice with Tris-buffered saline containing 0.05% Tween-20 (PBST). The cells were subsequently immobilized for 30 min, fixed with 4% paraformaldehyde at 25°C for 30 min and rinsed thrice with PBST. PBS containing 0.2% Triton X-100 was added to the plate for 10 min for permeabilization and the plate was subsequently rinsed thrice with PBST. The cells were incubated for 1 h at 25°C away from light with rhodamine phalloidin (PHDR1; 1:40 dilution; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and rinsed thrice with PBST. Cells were subsequently incubated for 15 min away from light with 5 µg/ml DAPI and rinsed thrice with PBST. Finally, the cells were observed and photographed under fluorescence microscope at ×1,000 magnification following the addition of anti-fade mounting medium (cat. no. P0126; Beyotime Institute of Biotechnology).

Cells in the logarithmic growth phase were grown to ~70% confluence in 6-well plates and treated with 0, 25, 50 and 100 µM myricetin for 48 h. Total RNA extracted using Trizol from each sample was used to determine concentration and purity and the rest of the RNA samples were subjected to reverse transcription using Prime Script RT Master Mix kit (TaKaRa Biotechnology Co., Ltd., Dalian, China). RNA was reverse-transcribed into cDNA using AceQ qPCR SYBR Green Master Mix (Vazyme, Piscataway, NJ, USA). A total of 3 wells were used for cDNA samples of each concentration and the experiments were repeated thrice. Primers used for real-time PCR analysis are presented in Table I. GAPDH was used as a reference gene. The thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min prior to 40 cycles at 95°C for 10 sec and at 60°C for 34 sec. Relative mRNA levels were evaluated by 2^−ΔΔCq method (18).

Western blot analysis was performed to determine the expression of E-cadherin, N-cadherin and vimentin proteins. The cells were grown to ~50% in 6-well plates and treated with 0, 25, 50 and 100 µM myricetin for 48 h. Cells were lysed by adding RIPA buffer (cat. no. P0013B; Biyun Biotechnology Co., Ltd.) subsequent to being harvested and the supernatant was collected. SDS-PAGE protein sample buffer (cat. no. P0015L; Biyun Biotechnology Co., Ltd) was added into the supernatant prior to protein sample preparation by boiling the aforementioned mixed liquid for 5–10 min in a water bath at 100°C. Simultaneously, the supernatant was collected to determine the concentration of protein samples by bicinchoninic acid protein assay. Protein samples (40 µg) were loaded onto SDS-PAGE (10% gels) and transferred to a polyvinylidene difluoride membrane. Membranes were blocked in 5% non-fat dried milk and incubated overnight at 4°C subsequent to adding diluted monoclonal antibody, including E-cadherin (1:1,000), N-cadherin (1:1,000), vimentin (1:1,000) and GAPDH (1:1,000). Subsequently, the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody [HRP-conjugated anti-mouse and anti-rabbit IgG (1:2,000)] was added to incubated for 2 h at 25°C. The chemiluminescence was determined using a gel imaging system and Pierce Enhanced Chemiluminescence Plus (Thermo Fisher Scientific, Inc.). The membranes were incubated with GAPDH antibody as a control and the intensity of the bands was quantified using ImageJ 1.42q software (National Institutes of Health, Bethesda, MD, USA).

The experimental data were analyzed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA) and Excel 2003 (Microsoft Corporation, Redmond, WA, USA) through one-way analysis of variance followed by Tukey's post hoc test for multiple comparisons and Student's t-test. All the experimental data were expressed as mean ± standard deviation The variance analysis was used to compare the differences between groups of measurement data. P<0.05 was considered to indicate a statistically significant difference.

Compared with the control group (0 µM), the viability of MHCC97H cells did not significantly change when cells were treated with 10–100 µM of myricetin. However, there was a significant decrease in viability at 200 µM (P<0.01; Fig. 1). This suggests that the cytotoxicity of myricetin at 0–100 µM decreased compared with that at 200 µM. Therefore, the concentrations of 25, 50 and 100 µM were selected to treat MHCC97H cells.

The cell scratch assay indicated that compared with the control group, the migration of MHCC97H cells was inhibited by 100 µM myricetin when the cells were treated for 24 and 48 h (Fig. 2A).

The Transwell migration assay indicated that, compared with the control group, an increase in drug concentration (50 and 100 µM) significantly decreased the number of migrated cells, and the invasive ability of MHCC97H cells was reduced in response to 100 µM myricetin (Fig. 2B). The relative cell count at the bottom of the membrane is indicated in Fig. 2C (P<0.01). Therefore, migration and invasion of MHCC97H cells may be inhibited in response to myricetin treatment.

RT-qPCR analysis indicated that the relative mRNA expression of E-cadherin in MHCC97H cells significantly increased at 25 µM myricetin (P<0.01; Fig. 3A) and the relative mRNA expression level of N-cadherin significantly decreased at 100 µM, along with that of vimentin (P<0.01; Fig. 3A). The aforementioned results suggest that myricetin may affect the mRNA expression level of genes associated with migration and invasion.

Western blotting indicated that the relative expression of E-cadherin in MHCC97H cells significantly increased (P<0.01; Fig. 3B and C) and the expression level of N-cadherin and vimentin protein significantly decreased (P<0.01; Fig. 3B and C). RT-qPCR and western blotting indicated that the migration and invasion of MHCC97H cells may be inhibited by myricetin treatment through the upregulation of E-cadherin and the downregulation of N-cadherin and vimentin.

The filamentous actin (F-actin) cytoskeleton serves an important role in cell migration and invasion (19). Compared with the control group, the number of fibers decreased in MHCC97H cells, and the filopodia and lamellipodia at the cell edge were also weakened with increased myricetin concentration (Fig. 4). The effects of myricetin on the migration and invasion of MHCC97H cells may be mediated by the rearrangement of F-actin cytoskeleton fibers.