Human HO8910PM and HO8910 EOC cell lines were obtained from the Chinese Academy of Sciences Cell Bank. The parental HO8910 cell line was established from ascites of a patient with malignant papillary serous adenocarcinoma of the ovary. HO8910PM is a highly metastatic clone obtained by limiting-dilution cloning of the HO8910 line and has been used in many studies, owing to its highly metastatic activity. Cells were grown in Roswell Park Memorial Institute (RPMI)-1640 medium (Thermo Scientific Hyclone, Logan, UT, USA) plus 10% fetal bovine serum (FBS; Gibco/Life Technologies, Carlsbad, CA, USA) at 37°C in a humidified atmosphere at normoxic conditions of 20% O₂, 5% CO₂ and 75% N₂. Experiments were performed in 6-well culture plates at 80% cell confluence.

3-Methyladenine (3-MA), a specific inhibitor of autophagy and inhibitor of the conversion of LC3-I to LC3-II (26–29), was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit polyclonal anti-LC3B antibody (no. 2775) and rabbit monoclonal anti-Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) antibody (no. 4035) were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal anti-β-actin antibody (mAb 8226) was purchased from Abcam (Cambridge, England, UK). Mouse monoclonal anti-ras homolog gene family, member A (RhoA) antibody (no. ARH03) and rhodamine phalloidin (no. PHDR1) were purchased from Cytoskeleton Inc. (Denver, CO, USA). The Rho Assay Reagent (Rhotekin RBD, agarose; recombinant protein expressed in Escherichia coli; no. 14-383, lot no. 2013105) was purchased from EMD Millipore (Billerica, MA, USA). Goat anti-rabbit IgG secondary antibody [AlexaFluor 488 goat anti-rabbit IgG (H+L); no. A11008] was purchased from Invitrogen/Life Technologies (Grand Island, NY, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Millipore.

LC3B target-specific small-interfering RNA (siRNA) plasmid vector and a scrambled siRNA control plasmid were purchased from Shanghai GenePharma, Ltd. (Shanghai, China). The siRNA sequences used were: LC3B, sense: 5′-GCGAGU UGGUCAAGAUCAUTT-3′ and antisense: 5′-AUGAUC UUGACCAACUCGCTT-3′; negative control, sense: 5′-UUC UCCGAACGUGUCACGUTT-3′ and antisense: 5′-ACGUGA CACGUUCGGAGAATT-3′. The cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen/Life Technologies) according to the manufacturer’s instructions. Cells (2×10⁵ per well) were seeded in 6-well culture plates at 30–50% confluence. Each plate was transfected with 100 pmol plasmid containing LC3B siRNA or negative control siRNA and 5 μl Lipofectamine 2000. Transfection efficiency averaged 60–70% as measured by green fluorescent protein expression. The cells were allowed to recover in RPMI-1640 medium for 6 h after transfection. Western blotting was performed to validate the knockdown efficiency, and the cells were divided for different assays.

Hypoxic environmental conditions were achieved by culturing cells in an airtight humidified chamber with a gas mixture containing 1% O₂, 5% CO₂ and 94% N₂. After 48 h, transfection with siRNA was performed or 3-MA was dissolved in phosphate-buffered saline, pH 7.4 (PBS) and added to the medium at a final concentration of 1.25, 2.5, or 5 mM. The flasks were exposed to hypoxic conditions or maintained in a normoxic environment for an additional 24 h, and the assays were performed.

Cells were collected for western blotting to determine the protein expression and trypan blue exclusion to determine cell viability. Briefly, proteins from the total cell lysates were separated by 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked in 5% non-fat dried milk, washed in Tris-buffered saline containing 0.1% Tween-20, and incubated at 4°C overnight with specific primary antibodies: Rabbit polyclonal anti-LC3B antibody (1:500 dilution), mouse monoclonal anti-RhoA antibody (1:1000 dilution), rabbit monoclonal anti-ROCK1 antibody (1:1000 dilution), and mouse monoclonal anti-β-actin antibody (1:5000 dilution) as a loading control. This was followed by incubation with HRP-conjugated secondary antibodies. The proteins were detected with an enhanced chemiluminescence detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).

Cell migration was assessed with a Transwell chamber system (Corning Inc., Life Sciences, Tewksbury, MA, USA). A total of 2.0×10⁵ cells in RPMI-1640 medium were added to the upper chamber under normoxic or hypoxic conditions. RPMI-1640 medium containing 10% FBS was added to the lower chamber. Migration assays were conducted for 6 h at 37°C. The insert was then washed with 1X PBS, and the non-migrated cells were removed with a cotton swab. The cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. Excess staining was washed away with water. The cells were identified under a microscope, and a cell count of five different fields was performed.

Cell invasion was assessed with Matrigel-coated 24-well chambers. A total of 1.0×10⁵ cells in RPMI-1640 medium were added to the insert. RPMI-1640 medium containing 10% FBS was added to the lower chamber. Invasion assays were conducted for 24 h at 37°C under normoxic or hypoxic conditions, and the cells were fixed and counted as described for cell migration assays. For the invasion assays involving siRNA, the cells were transfected with LC3B siRNA for 48 h prior to invasion assays being performed.

Cell adhesion was assessed with Matrigel-coated 96-well plates [dry-coated with Matrigel and blocked with 1% bovine serum albumin (BSA) for 1 h]. A total of 4×10⁴ cells were allowed to adhere to Matrigel-coated wells for 2 h at 37°C under hypoxic conditions. The wells were washed three times with PBS. A volume of 10 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) substrate was added to each well, and incubation was continued for an additional 4 h. The number of adherent cells in each well was quantified by staining with dimethyl sulfoxide and absorbance was measured at 490 nm.

Control cells or cells subjected to LC3B knockdown were washed with PBS and blocked with 1% BSA. The cells were then incubated with rabbit polyclonal anti-LC3B antibody (1:500 dilution) at 4°C overnight. The cells were washed with PBS, incubated with AlexaFluor 488 goat anti-rabbit IgG (H+L) (1:300 dilution) for 2 h, and rhodamine phalloidin (1:1000 dilution) was added for the last 30 min. Cell nuclei were stained during the last 8 min with 20 μl 4′,6-diamidino-2-phenylindole (DAPI). Images were captured by laser confocal microscopy (Zeiss LSM 710), and image analysis was performed with ZEN microscopic image analysis software (Zeiss).

Activated RhoA in cells was detected with Rho Assay Reagent according to the manufacturer’s instructions. Activated GTP-RhoA was detected by western blotting and mouse monoclonal anti-RhoA antibody (1:1000 dilution).

Experiments were repeated at least three times. Continuous data are presented as mean ± standard deviation. To assess differences among groups, One-way analysis of variance was performed, followed by the Tukey and Dunnett T3 post-hoc tests. P<0.05 was considered statistically significant. All the statistical analyses were two-sided and performed with SPSS software (version 15.0; SPSS Inc., Chicago, IL, USA).

The results showed that hypoxic conditions promoted HO8910PM and HO8910 cell migration, invasion and adhesion (Figs. 1–3, respectively). When LC3B was blocked with 3-MA or LC3B siRNA treatment, cell migration and invasion were significantly decreased compared to that in control cells (1% O₂/hypoxia alone; all p<0.001 for both HO8910PM and HO8910 cells). In addition, the cells treated with LC3B siRNA showed significantly less migration (p=0.021 in HO8910PM cells and p<0.001 in HO8910 cells) (Fig. 1) and invasion (p<0.001 for the two cell types) (Fig. 2) compared to that in 1.25 mM 3-MA-treated cells. Adhesion was significantly reduced in cells treated with LC3B siRNA compared to that in 1.25 mM 3-MA-treated cells (p=0.021 in HO8910PM cells and p=0.004 in HO8910 cells) (Fig. 3).

LC3B expression and conversion of LC3B-I to LC3B-II was observed in the two cell lines under hypoxic conditions (Figs. 4 and 5). In addition, RhoA expression was clearly increased under hypoxic conditions. LC3B siRNA (Fig. 5) and 24-h 3-MA treatment (Figs. 4 and 5) each decreased LC3B expression under hypoxic conditions. Control siRNA had no effect on LC3B expression. We also found that 3-MA treatment decreased activated RhoA expression and expression of the downstream effector molecule ROCK1 in HO8910 cells, and LC3B siRNA decreased activated RhoA and ROCK1 expression in HO8910PM cells. We also confirmed hypoxia-induced RhoA expression, which was inhibited via LC3B siRNA in the two cell lines, though it appeared that control siRNA had some interference effect on RhoA expression in HO8910 cells. These results suggested that RhoA may be associated with LC3B and that the mechanism by which LC3B promotes metastasis may involve the RhoA pathway in EOC cells.

Filamentous actin (F-actin) and LC3B in EOCs under hypoxic conditions were detected by laser scanning confocal microscopy (Fig. 6). The cells transfected with LC3 siRNA showed F-actin fibers that lacked tension and direction and showed a disordered arrangement. The cells transfected with control siRNA showed F-actin fibers with good tension and direction and filament bundles, suggestive of good invasion ability.