The HONE-1 and 293T cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco-BRL (Invitrogen Life Technologies, Inc., Carlsbad, CA, USA) and fetal bovine serum (FBS) was purchased from Sijiqing Biotech. Co. (Hangzhou, China). TRIzol reagent was purchased from Invitrogen Life Technologies, Inc. and a Cell Counting kit-8 (CCK-8) was purchased from Dojindo Biochem (Shanghai, China). An Annexin V/fluorescein isothiocyanate (FITC) kit and Matrigel were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Crystal violet, Giemsa, trypsinase, acrylamide, SDS and phosphate-buffered saline (PBS) were purchased from JRDun Biotech (Shanghai, China). Transwell well culture chambers were purchased from Corning (Corning, NY, USA). A bicinchoninic acid (BCA) protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA) and nitrocellulose filter membranes were purchased from Millipore (Billerica, MA, USA). Antibodies against caspase-3 (cat. no. Ab32351; rabbit monoclonal; 1:1,000), HMGB1 (cat. no. Ab79823; rabbit monoclonal; 1:800) and advanced glycation end products (RAGE; cat. no. Ab3611; rabbit polyclonal; 1:1,000) were purchased from Abcam (Cambridge, MA, USA); monoclonal antibodies against extracellular signal-regulated kinase (ERK)1/2 (cat. no. 9102; rabbit polyclonal; 1:1,000), phosphorylated (p)-ERK1/2 (cat. no. 4376; rabbit IgG; 1:1,000) and GAPDH (cat. no. 5174; rabbit monoclonal; 1:2,000) were purchased from CST Biotech (Shanghai, China); monoclonal antibodies against B-cell lymphoma 2 (Bcl-2; cat. no. Sc-492; rabbit polyclonal; 1:400) and Bcl-2-associated X protein (Bax; cat. no. Sc-493; rabbit IgG; 1:500) were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX, USA). Goat-anti-rabbit/rat horseradish peroxidase-conjugated secondary antibodies were purchased from Beyotime Institute of Biotechnology (Haimen, China).

HONE-1 cells were cultured in DMEM containing 15% (v/v) heat-inactivated FBS, 100 mg/ml streptomycin (Corning Incorporated, Corning, NY, USA) and 100 U/ml penicillin (Corning Incorporated) at 37°C in a humidified atmosphere containing 5% CO₂.

HMGB1-specific small interfering (si)RNA overexpression vector was constructed for RNA interference as previously described (17). The resulting recombinant lentiviruses were transfected into HONE-1 cells to inhibit the expression of HMGB1. The siRNA target sequence was as follows: 5′-TGGTGATGTTGCGAAGAAA-3′. HMGB1 expression in untreated HONE-1 cells, cells treated with control vector (Mock group), and HONE-1 cells with HMGB1 knockdown (HMGB1-KD) was detected using real-time fluorogenic reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis.

HONE-1 cells were harvested and total RNA was extracted using TRIzol according to the manufacturer's instructions. Total RNA was used for cDNA synthesis of HMGB1 and GAPDH by reverse transcription using an ABI-7300 real-time PCR apparatus (Applied Biosystems, Thermo Fisher Scientific). All mRNA primers were designed using Primer Premier 5.0 software (Premier Biosoft, Palo Alto, CA, USA) and synthesized by JRDun Biotech. Primers used in for PCR are listed in Table I. qPCR was performed according to the manufacturer's instructions of the quantitative real-time RT-PCR reaction kit (SYBR Green; Thermo Fisher Scientific). Thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, and 60°C for 45 sec. Gene expression levels were quantified using the ΔΔCt method.

HONE-1 cells were harvested and total protein was extracted using radioimmunoprecipitation acid lysis buffer (JRDUN Biotechechnology Co., Ltd., Shanghai, China). The protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein (20 µg) were separated by 10 or 15% SDS/PAGE, electrotransferred onto a nitrocellulose filter membrane and probed with various primary antibodies overnight at 4°C, followed by detection with horseradish-peroxidase-conjugated secondary antibodies and electrochemiluminescence development (Millipore) with X-ray film (Kodak, Rochester, NY, USA) exposure. To normalize for protein loading, antibodies directed against GAPDH were used, and the proteins expression levels were expressed as a relative value to that of GAPDH.

Cells (5×10³/100 µl) were seeded in 96-well plates and cultured for 0, 12, 24 or 48 h at 37°C. Subsequently, 100 µl serum-free DMEM containing 10% CCK-8 reagent (v/v) was added to each well, and cells were cultured for 1 h at 37°C. Finally, optical density values (OD) were determined at 450 nm using a 96-well plate reader (Multiskan MK3; Thermo Fisher Scientific) (18).

HONE-1 cells (5×10⁴ per group treated as above) were harvested, washed with PBS and stained using the Annexin V/FITC kit according to the manufacturer's instructions. Apoptosis was detected using a FACScalibur flow cytometer (BD Biosciences). The percentage of cells in early apoptosis was the population of Annexin V-positive and PI-negative cells, while the percentage of cells in late apoptosis was resembled by Annexin V-positive and PI-positive cells (19).

A wound-healing assay was performed according to a previously reported method with minor modifications (20,21). HONE-1 cells were treated with control lentivirus (Mock group) or HMGB1 siRNA lentivirus (HMGB1-KD), and seeded onto 35-mm² petri dishes, and a sterile pipette (200 µl) was used to scratch the cell monolayer to generate a line-shaped wound. Cells were washed once with cold PBS, and incubated for 48 h. Migration of the cells was evaluated at 0, 24, 48 and 72 h.

A cell adhesion assay was performed in 12-well plates according to a previous method with minor modification (22,23). The wells were pre-coated with fibronectin (BD Biosciences, Franklin Lakes, NJ, USA) overnight at room temperature. HONE-1 cells were harvested and re-suspended in DMEM containing 10% FBS. Subsequently 1×10⁵ cells were seeded into each well and incubated at 37°C for 1 h. The wells were washed twice with warm PBS to remove the unattached cells, and the attached cells were then stained with Giemsa for 10 min. The cells were observed by using an optical Olympus IX50 microscope (Olympus, Tokyo, Japan).

The invasive abilities of HONE-1 cells were evaluated according to a previously described method with minor modifications (24). Cell invasion assays were performed using Transwell culture chambers, which were pre-coated with 80 ml Matrigel. The coated filters were thoroughly washed with PBS and dried immediately prior to use. 0.75 ml DMEM containing 10% FBS was placed in the lower chamber, while 0.5 ml cell suspension (1×10⁵/ml) in DMEM containing 1% FBS was placed in the upper chamber followed by incubation for 48 h at 37°C in an atmosphere containing 5% CO₂. The number of the cells which had transgressed through the Matrigel-coated filter was evaluated by counting cells stained with 0.5% crystal violet solution under an optical microscope (Olympus, Tokyo Japan).

Values are expressed as the mean ± standard deviation, and statistical analyses were performed via a one-way analysis of variance followed by post-hoc Dunnett's test. SPSS software version 18.0 (International Business Machines, Armonk, NY, USA) was used for statistical analysis. P<0.05 was considered to indicate a statistically significant difference between values.

As shown in Fig. 1A, the expression of HMGB1 was significantly down-regulated in HONE-1 cells transfected with HMGB1 siRNA expression plasmid, compared to that in the untreated and mock-transfected groups (P<0.01). Furthermore, western blot analysis demonstrated that the protein expression of HMGB1 was obviously decreased after transfection, which indicated the successful construction of HONE-1 cells with HMGB1 knockdown (Fig. 1B).

HONE-1 cell proliferation was detected using the CCK-8 assay after transfection. As shown in Table II, no significant difference in proliferation was observed between the untreated and mock-transfected HONE-1 cells (P>0.05). However, the proliferation of HONE-1 cells with HMGB1 knockdown was significantly inhibited at 12, 24 and 48 h (P<0.01), compared to that of the untreated and mock-transfected HONE-1 cells. Therefore, the results of the present study indicated that HMGB1 knockdown significantly inhibited the proliferation of HONE-1 cells.

To assess whether the anti-proliferative effects of HMGB1 knockdown on HONE-1 cells were due to induction of apoptosis, flow cytometric analysis following staining with PI and Annexin V-FITC was performed. As shown in Fig. 2, the apoptotic rate in the HMGB1-knockdown group was 42.4%, which was significantly higher than that in untreated (6.2%) and in the mock-transfected HONE-1 cells (7.8%). This result revealed that HMGB1 knockdown significantly induced apoptosis in HONE-1 cells.

As demonstrated by the wound healing assay, HMGB1 knockdown effectively inhibited the migration of HONE-1 cells at 24, 48 and 72 h (Fig. 3). In the untreated and mock-transfected HONE-1 cells, the wounds were obviously smaller than those in the HMGB1 knockdown group at all of the observed time-points and, by contrast to the HMGB1 knockdown group, the wounds had disappeared in the untreated and mock-transfected groups after 72 h. Furthermore, as indicated by the Transwell assay, HMGB1 knockdown effectively inhibited the invasion of HONE-1 cells (Fig. 4). As shown in Fig. 5, there was no obvious difference in cell adhesion between the untreated and mock-transfected HONE-1 cells. However, in the HMGB1 knockdown group, cell adhesion was significantly inhibited compared to that in the untreated and mock-transfected HONE-1 cells. The combined results of the wound healing, Transwell invasion and cell adhesion assay indicated that HMGB1 knockdown significantly suppressed the metastasis-forming capacity of HONE-1 cells.

The results of the present study indicated that HMGB1 knockdown inhibited the proliferation, migration and invasion of HONE-1 cells, while enhancing their apoptotic rate. To investigate the possible underlying mechanisms, the expression of apoptosis-associated signaling and effector proteins was determined by western blot analysis. As shown in Fig. 6, there was no obviously difference in the expression of Bcl-2, Bax, cleaved caspase-3, RAGE, p-ERK1/2 and ERK1/2 between the untreated and mock-transfected HONE-1 cells. Western blotting was repeated three times and similar results were obtained. However, in the HMGB1-knockdown group, the expression of Bcl-2, RAGE and p-ERK1/2 was down-regulated, whereas the expression of caspase-3 and Bax was obviously upregulated compared to that in the untreated and mock-transfected HONE-1 cells. These results indicated that the mechanisms of the inhibitory effects of HMGB1 knockdown on HONE-1 cells may be associated with the induction of apoptosis via the HMGB1/RAGE pathway.