The SK-ES-1 and RD-ES human ES cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). RPMI-1640 medium, fetal bovine serum (FBS), PBS, dimethyl sulfoxide and Cell Counting Kit-8 (CCK-8) were provided by Beijing Transgen Biotech Co., Ltd. (Beijing, China). Antibodies against Beclin-1 (ab207612, 1:1,000), matrix metalloproteinase (MMP)-2 (ab92536, 1:1,000), MMP-9 (ab194314, 1:1,000) and β-actin (ab8227, 1:1,000) were all purchased from Abcam (Cambridge, UK). Goat anti-rabbit IgG (H+L), horseradish peroxidase-conjugated secondary antibodies (HS101-01, 1:2,000) were both purchased from Beijing Transgen Biotech Co., Ltd. Lipofectamine^® 2000 and OPTI-MEM were both purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Matrigel was purchased from BD Biosciences (San Jose, CA, USA). The Transwell invasion chambers were purchased from Costar (Cambridge, MA, USA). Crystal violet staining solution was purchased from Beyotime Institute of Biotechnology (Haimen, China). si-Beclin-1 against the BECLIN-1 gene (NM_003766) and control siRNA (si-CON) were constructed by Shanghai GeneChem Co., Ltd. (Shanghai, China).

SK-ES-1 and RD-ES cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated in a humidified atmosphere containing 5% CO₂ at 37°C. All cells used in the present study were subjected to <20 cell passages. SK-ES-1 cells at logarithmic phase were seeded at a density of 3×10⁵ cells/well in a 6-well plate for 24 h prior to transfection. Lipofectamine 2000 (10 µl diluted in 250 µl OPTI-MEM) was used for the transfection of 4 µg si-Beclin-1 or empty vector diluted in 250 µl OPTI-MEM, followed by incubation of the samples for 20 min at room temperature. The plasmid DNA-Lipofectamine 2000 complex was then added into 500 µl OPTI-MEM and incubated at 37°C with 5% CO₂ in an incubator for 6 h. Subsequently, the medium was replaced and the cells were incubated for 24 or 48 h in RPMI-1640 medium supplemented with 10% FBS prior to use in the corresponding experiments, which included a blank control group (non-transfected SK-ES-1 cells), a negative control group (SK-ES-1 cells transfected with blank plasmid, i.e., si-CON) and an experimental group (SK-ES-1 cells transfected with si-Beclin-1). Transfected cells were collected at 24 or 48 h post-transfection and used in subsequent experiments.

Cell growth was determined using the CCK-8 assay. In brief, cells infected with si-Beclin-1 or si-CON and non-transfected SK-ES-1 cells were incubated in 96-well plates at a density of 3×10³ cells/well. Cells were treated with 10 µl CCK-8 reagent at 24, 48 and 72 h and then measured at 450 nm using a Universal Microplate reader (EL800; Bio-Tek Instruments Inc., Winooski, VT, USA).

The invasive capacity of SK-ES-1 cells was detected via Matrigel-coated Transwell cell culture chambers (8 µm pore size). Following transfection for 24 h, SK-ES-1 cells of the three different groups were collected and suspended in serum-free medium. Isolated cells were then added to the upper chamber of the Transwell insert at a density of 4×10⁴ cells/well and the lower wells were filled with complete growth medium supplemented with 10% FBS. All samples were incubated for 24 h in a CO₂ incubator. Non-invading cells (on the upper membrane surface) were removed using a cotton swab and invading cells (on the lower membrane surface) were fixed with 95% ethanol for 15 min at 25°C, stained with 0.1% crystal violet staining solution for 20 min at 25°C, then counted under a phase-contrast microscope in three random fields (magnification, ×200).

Migration of SK-ES-1 cells was measured using wound healing assays. After 24 h of transfection, SK-ES-1 cells of the three different groups were seeded at a density of 5×10⁵ cells/well in a 6-well culture plate to form a confluent monolayer. Cells were wounded with a sterile 100 µl pipette tip. All cells in the plates were incubated in fresh RPMI-1640 medium with 10% FBS for 24 h. Then scratch wounds were observed using a phase-contrast microscope and images were captured of each wound.

Following transfection, SK-ES-1 cells of the three different groups were seeded in 6-well plates at a concentration of 3×10⁵ cells/well and incubated in RPMI-1640 medium with 10% FBS for 48 h. The cells were collected and lysed in radioimmunoprecipitation assay buffer containing phenylmethane sulfonyl fluoride and phosphatase inhibitor cocktail (Sigma Aldrich; Merck KGaA, Darmstadt, Germany). Each sample was centrifuged at 17,105.6 × g for 10 min at 4°C using a Universal 320R centrifuge (Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany), to remove cell debris and collect the supernatant for immunoblotting. Protein concentrations were calculated using a bicinchoninic acid assay kit (Beijing Transgen Biotech Co., Ltd.) according to the manufacturer's instructions with bovine serum albumin as the relative standard. Proteins (10 µl) were loaded and separated using SDS-PAGE (10% gel, 100 V for 2 h under reducing conditions). Following electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes in a tris-glycine transfer buffer and incubated with antibodies against β-actin, Beclin-1, MMP-2 and MMP-9 overnight at 4°C. The PVDF membranes were washed in Tris-buffered saline Tween-20 (TBST) three times. Secondary HRP-conjugated antibodies were added at 1:2,000 dilution and incubated for 2 h at 25°C. The PVDF membranes were washed a further three times in TBST. Immunoreactive proteins were detected using an enhanced chemiluminescence system (GE Healthcare, Chicago, IL, USA) according to the manufacturer's instructions followed by exposure to X-ray films. Western blotting data was quantified using ImageJ software (version 7.0; National Institutes of Health, Bethesda, MD, USA).

Data were analyzed using the SPSS package for Windows (version 19.0; IBM Corp., Armonk, NY, USA). Quantitative data are expressed as mean ± standard deviation. Statistical analysis was performed using a one-way analysis of variance with the Student-Newman-Keuls method as a post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Endogenous expression of Beclin-1 in the human ES SK-ES-1 and RD-ES cell lines was evaluated using western blot analysis. As presented in Fig. 1A and B, expression of Beclin-1 was significantly increased in the SK-ES-1 cell line compared with the RD-ES cell line (P<0.05). Thus, the SK-ES-1 cell line was used for Beclin-1 knockdown.

Protein levels of Beclin-1 were determined using western blotting following transfection of SK-ES-1 cells with si-Beclin-1 or si-CON vectors for 48 h. As presented in Fig. 1C and D, Beclin-1 expression was significantly decreased in the si-Beclin-1 group compared with the blank control group (P<0.01)

The effect of Beclin-1 knockdown on SK-ES-1 cell growth was determined using a CCK-8 assay. As presented in Fig. 2A, knockdown of Beclin-1 significantly suppressed the growth of SK-ES-1 cells (P<0.05 at 24 h and P<0.01 at 48 h).

Transwell and wound healing assays were conducted to confirm the effect of Beclin-1 knockdown on the invasion and migration of SK-ES-1 cells. Representative micrographs of Transwell filters are presented in Fig. 2B. The invasive cell count, also presented in Fig. 2B, demonstrated that the invasive potential was significantly decreased in the si-Beclin-1 group relative to the blank control group (P<0.01). Furthermore, Beclin-1 knockdown resulted in a decrease in migration capability, as presented in Fig. 2C and D (P<0.01).

A western blot assay was performed to investigate the effect of Beclin-1 knockdown on the expression of MMP-2 and MMP-9, since it is generally acknowledged that they serve functions in tumor invasion, and metastasis (12–14). As presented in Fig. 3A and B, MMP-9 expression was significantly decreased in the si-Beclin-1 group compared with the Con group (P<0.01); however, no significant difference in the expression of MMP-2 was observed between the two groups. These results suggest that Beclin-1 knockdown may inhibit invasion and metastasis of SK-ES-1 cells via downregulation of the expression of MMP-9.