Forty pairs of ESCC and healthy adjacent esophageal tissue samples were obtained from surgically excised specimens of ESCC from patients at the Affiliated Cancer Hospital of Xinjiang Medical University (Ürümqi, China) between July and December 2013. The tumor and adjacent healthy tissues were frozen in liquid nitrogen immediately following resection. The patients in the current study had not received chemotherapy or radiation therapy prior to surgery. The present study was approved by the Ethics Committee of the Affiliated Cancer Hospital of Xinjiang Medical University. Written informed consent was provided by the families of all of the patients.

The Eca109 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Zhejiang Tianhang Biological Technology Co., Ltd., Zhejiang, China), penicillin (100 U/ml) and streptomycin (100 mg/ml) (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified 5% CO₂ atmosphere.

The KYSE150 cell line was purchased from the Beijing Institute of Cancer (Beijing, China). The cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37°C in a humidified 5% CO₂ atmosphere.

The LV3 vector was purchased from Shanghai GenePharma Co., Ltd., Shanghai, China). The small hairpin RNA (shRNA) targeting sequences for survivin and the control are presented in Table I. The sequences were inserted between the HindIII and XhoI sites of the LV3 vector, which yielded LV3-survivin shRNA and LV3-control shRNA plasmids (synthesized by Shanghai GenePharma Co. Ltd.). The constructs were verified by DNA sequence analysis.

The GV142 plasmid was purchased from GeneChem Co., Ltd. (Shanghai, China). For the GV142-survivin overexpression and GV142-control plasmid construction, GV227 (GeneChem Co., Ltd.) was used as the template, and the survivin and control polymerase chain reaction (PCR) primers used are presented in Table II. The resulting PCR products were inserted into the GV142 vector between HindIII and XhoI sites, yielding GV142-survivin overexpression and GV142-control plasmids.

Prior to transfection, 2×10⁵ cells/well were placed into 6-well plates cultivated in serum-free culture medium and antibiotics, and grown overnight until they reached 70–80% confluence. Plasmid transfection was performed using Lipofectamine™ 2000 transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The plasmids and Lipofectamine™ 2000 were diluted in Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific, Inc.) separately and incubated for 10 min at room temperature. The diluted solutions were mixed and incubated for 20 min at room temperature. Subsequently, the mixtures were added to each well containing cells and medium. The cells with only the transfection reagent served as a blank control. Cell culture plates were incubated for 6 h at 37°C in a CO₂ incubator. Culture medium containing 10% FBS was added and cells were incubated under the above-mentioned conditions.

Cells were seeded in 96-well plates at a density of 5×10³ cells/well in a volume of 100 μl culture medium per well. After 24 h, cells were exposed to survivin inhibitor, YM155 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at various concentrations (0, 0.005, 0.05, 0.5, 5 and 50 μM) for 48 h. Following incubation at 37°C, 0.4% Trypan Blue solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. Viability was assessed by counting the relative number of live, unstained cells to total cells, and the half-inhibitory concentration (IC₅₀) for YM155 was derived from a logarithmic plot.

Cells treated with YM155 (concentrations at, above and below the IC₅₀) were harvested at 48 h, and the total RNA and protein was extracted to determine the expression levels of survivin, IKKα, IKKβ and NF-κB p65 by reverse transcription quantitative (RT-q) PCR and western blotting.

At 48 h post-transfection, total cellular RNA was extracted using TRIzol (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from 1 μg total RNA using Maloney Murine Leukemia Virus Reverse Transcriptase (Promega Corporation, Madison, WI, USA) and an oligo-deoxy-thymine nucleotide primer (Promega Corporation) according to the manufacturer's protocols. DNA content was measured using a UV/visible spectrophotometer (Ultrospec 2000; GE Healthcare Life Sciences, Chalfont, UK). Primer sequences are presented in Table III. Primers used in the current study were synthesized by Shanghai Shenggong Biology Engineering Technology Service, Ltd. (Shanghai, China). RT-qPCR assays used the TaqMan^® Fast Virus 1-Step Master mix kit (Thermo Fisher Scientific, Inc.). The reaction system was: 12 μl SYBR^® green reagent (Thermo Fisher Scientific, Inc.), 0.2 μM each primer, 1 μl cDNA template, and 6 μl nuclease-free distilled water (Thermo Fisher Scientific, Inc.). GAPDH served as an internal standard to evaluate the relative expression levels of the target genes. qPCR analysis was performed on an Applied Biosystems^® 7500 Fast Real Time PCR instrument (Thermo Fisher Scientific, Inc.). The PCR conditions, performed for 40 cycles, were as follows: 2 min at 50°C, 2 min at 95°C, 15 sec at 95°C, 15 sec at 55–60°C, 1 min at 72°C. The relative quantification transcript levels were determined using the 2^−ΔΔCq method. Specificities of all PCR amplifications were confirmed by melting curve analysis. All experiments were performed in triplicate and results are presented as the mean ± standard deviation.

At 48 h post-transfection, total protein was extracted, by washing twice with ice-cold phosphate-buffered saline (PBS; Thermo Fisher Scientific, Inc.), and resuspended in lysis buffer [1 M Tris HCl (pH 7.4), 5M NaCl, 0.5 M ethylene glycol tetraacetic acid, 0.5 M EDTA, NP-40, 10% SDS (Wuhan Boster Biological Technology, Ltd., Wuhan, China), glycerine, 10 μg/μl aprotinin, 10 μg/μl leupeptin, 10 μg/μl Pepstatin A, 10 mM phenylmethylsulfonyl fluoride, double-distilled H₂O] for 30 min on ice. Suspensions were centrifuged at 18,407 × g for 15 min at 4°C. The supernatant containing the protein was collected and the protein concentration of each lysate was determined by Pierce BCA protein assay (Thermo Fisher Scientific, Inc.). Protein (20 μg) was loaded for each sample. Proteins were denatured, subjected to SDS-PAGE using 10–15% polyacrylamide gels (Wuhan Boster Biological Technology, Ltd.), electrophoresed (stacking gel, 60 V for 45 min; separating gel, 100 V for 90 min) and electrophoretically transferred onto nitrocellulose membranes (Whatman; GE Healthcare Life Sciences). The membranes were blocked with 5% non-fat dried milk (Sigma-Aldrich) in Tris-buffered saline and Tween-20 (TBST; Beijing Solarbio Science & Technology Co., Ltd.,Beijing,China) at room temperature for 2 h. The membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit polyclonal anti-GAPDH (1:400; cat. no. BA2913; Wuhan Boster Biological Technology, Ltd.), which served as a loading control; rabbit polyclonal anti-survivin (1:1,000; cat. no. sc-10811; Santa Cruz Biotechnology Inc.); rabbit polyclonal anti-phosphorylated (p)-NF-κB p65 (pSer⁵³⁶) (1:1,000; cat. no. AF2006; Affinity Biosciences, Cell Signal Transduction, Cincinnati, OH, USA); rabbit polyclonal anti-NF-κB p65 (1:400; cat. no. BA0610; Wuhan Boster Biological Technology, Ltd.); rabbit polyclonal anti-IKKα (1:400; cat. no. BA1594-2; Wuhan Boster Biological Technology, Ltd.); and rabbit polyclonal anti-IKKβ (1:400; cat. no. BA4458-2; Wuhan Boster Biological Technology, Ltd.) were blocked in TBST. Membranes were washed three times (10 min per wash) with TBST at room temperature. Subsequently, the membranes were incubated with appropriate horseradish peroxidase-linked goat anti-rabbit secondary antibodies at a dilution of 1:1,000 (cat. no. BA1054; Wuhan Boster Biological Technology, Ltd.) diluted in TBST for 1 h at room temperature. Protein bands were visualized using an Enhanced Chemiluminescence Detection kit (Thermo Fisher Scientific, Inc.).

The human IKKβ promoter sequences (3,000 bp upstream) and random control sequence (size, 3,000 bp) were obtained by PCR amplification and inserted between the HindIII and XhoI sites of the pGL3 vector (Promega Corporation) yielding pGL3-IKKβ and pGL3-random control plasmids (synthesized by GeneChem Co., Ltd.). Eca109 and KYSE150 cells were seeded at 5×10⁵ cells per well in 6-well dishes one day prior to transfection. The cells were co-transfected with 0.1 μg pGL3-random control, pGL3-IKKβ or pGL3-basic firefly luciferase reporter construct, 0.01 μg pRL-TK Renilla luciferase reporter plasmid and the GV142-survivin overexpression plasmid, using Lipofectamine™ 2000. pRL-TK Renilla luciferase reporter plasmid was co-transfected to assess the transfection efficiency. Post-transfection (48 h), cells were harvested and lysed with 1X lysis buffer (Promega Corporation). Cell extracts (20 μl) were assayed for luciferase activity using the Dual-luciferase Reporter assay system kit (Promega Corporation) according to the manufacturer's protocols. Relative levels of reporter gene expression were expressed as ratios of firefly luciferase activity to Renilla luciferase (LU/RL). All experiments were performed in triplicate.

ChIP assays were performed using the EZ ChIP™ kit (EMD Millipore, Billerica, MA, USA) according to the manufacturer's protocols. Eca109 and KYSE150 cells were transfected with the GV142-survivin overexpression plasmid and fixed with 1% formaldehyde (Sigma-Aldrich) for 10 min. The Eca109 and KYSE150 cells were washed twice with 1X PBS, lysed, and sonicated to reduce DNA lengths to within the range of 200–1,000 bp. The survivin/DNA complexes were incubated with 4 μg rabbit antibody against survivin, 1 μl Normal Mouse IgG (dilution, 1:1,000), which served as the negative control, and 1 μl Anti-RNA Polymerase II (dilution, 1:1,000; both included in the EZ-ChIP™ kit) served as the positive control. The mixes were incubated at room temperature for 60–90 min. The immune complexes were precipitated, eluted, reverse-crosslinked and treated with proteinase K [Tiangen Biotech (Beijing) Co.,Ltd., Beijing, China]. The resulting DNA samples were amplified using primers for the putative survivin site in the human IKKα and IKKβ promoter region. The primer sequences are presented in Table IV. PCR fragments were separated and visualized on 1.8% agarose gels stained with ethidium bromide (Shanghai Bioleaf Biotech Co., Ltd., Shanghai, China). The ratios of the PCR products of survivin (IKKα, IKKβ and NF-κB p65) to GAPDH were used to determine the expression levels of target genes.

For cell cycle analysis, DNA labeling was conducted using the Cycle Test Plus DNA reagent kit (BestBio Co., Ltd., Shanghai, China). Labeling with propidium iodide (PI) and Annexin V was performed using an Annexin V staining kit (BestBio Co. Ltd.) for the detection of apoptotic cells and the assays were performed according to the manufacturer's protocols. Eca109 and KYSE150 cells were directly incubated, at 37°C for 48 h, in 6-well plates and collected 48 h following transfection. For the cell cycle analysis, the cells were washed with PBS for 5 min and subsequently centrifugation at 900 × g. The cells were collected and fixed in ice-cold 70% ethanol (Saihongrui Biotechnology Co., Ltd., Nanjing, China) for a minimum of 2 h at 4°C, followed by treatment with 0.2 mg/ml RNase A (EMD Millipore) in PBS for 30 min at 37°C. PI was added (final concentration, 25 μg/ml) and the cells were incubated for 30 min at 4°C in the dark. Analysis of the samples was conducted within 24 h. For the apoptosis assay, the transfected cells were washed twice with ice-cold PBS, and resuspended in 195 μl 1X Binding Buffer (EMD Millipore) to a concentration of 1×10⁴ cells/ml. Annexin V (5 μl) and PI were gently mixed with the cells and incubated for 15 min at room temperature in the dark. The dyes were washed out by centrifugation for 5 min at 94 × g and the cells were resuspended in 190 μl 1X Binding Buffer. PI staining solution (10 μl) was gently mixed in and incubated on ice and in the dark. The samples were analyzed within 1 h. All samples for the two assays consisted of 10,000 cells and were analyzed by fluorescence-activated cell sorting with a BD FACSMicroCount™ system (BD Biosciences, Franklin Lakes, NJ, USA).

SPSS version 17.0 for Windows (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. All data were expressed as the mean ± standard deviation from three experiments. The two-tailed Student's t-test was used to analyze the difference between groups and Fisher's exact test was used to analyze correlation between groups. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of survivin, IKKα, IKKβ and NF-κB p65 were evaluated by RT-qPCR in 40 paired ESCC and healthy tissue samples, as presented in Table V. In the present study, the expression of survivin was observed to be positively correlated with IKKα (r =0.370; P<0.05) and IKKβ mRNA expression levels (r =0.341; P<0.05) in ESCC samples.

Cells transfected with LV3-survivin shRNA and LV3-control shRNA plasmids were designated the survivin knockdown group and control group, respectively. The cells cultured with the transfection reagent only were considered as a blank group. At 48 h after transfection, cells were harvested for RT-qPCR and western blotting assays. The RT-qPCR analysis (Fig. 1) demonstrated that expression levels of survivin, NF-κB p65, IKKα and IKKβ were significantly reduced in the survivin knockdown group, compared with the control and blank group in Eca109 (Fig. 1A) and KYSE150 cells (Fig. 1C). Western blotting analysis also demonstrated that protein expression levels of survivin, the p-NF-κB p65, IKKα and IKKβ expression levels were similarly reduced in the survivin knockdown group of the two cell lines (Fig. 1B and D).

YM155, an inhibitor of survivin, was used to investigate the effect of survivin on cell viability, as well as the interaction between survivin and NF-κB p65, IKKα, and IKKβ expression levels in Eca109 and KYSE150 cells. YM155 was administered at concentrations from 0.005 to 50 μM, which significantly reduced cell viability in a dose-dependent manner in the Eca109 and KYSE150 cells at 48 h following administration. According to the logarithmic curve, the IC₅₀ of YM155 for Eca109 and KYSE150 cells was ~0.5 μM (Fig. 2A). Concentrations at, above, and below the IC₅₀ (0.05, 0.5 and 5 μM) were selected for further experiments.

Eca109 and KYSE150 cells were treated with YM155 at the selected concentrations for 48 h, and the expression levels of survivin and NF-κB p65, IKKα and IKKβ were measured by RT-qPCR and western blotting assays. The results demonstrated that YM155 effectively inhibited mRNA expression levels of survivin and NF-κB p65, IKKα and IKKβ, and protein expression levels of survivin and the p-NF-κB p65, IKKα and IKKβ in Eca109 (Fig. 2B and D) and KYSE150 (Fig. 2C and E) cells, which was comparable with the survivin shRNA knockdown.

Cells transfected with GV142-survivin overexpression plasmid and GV142-control plasmid were designated the survivin overexpression group and the control group, respectively. The cells with the transfection reagent only served as the blank group. At 48 h after transfection, cells were harvested for RT-qPCR and western blotting assays. RT-qPCR analysis demonstrated increased mRNA expression levels of survivin, NF-κB p65, IKKα and IKKβ in the survivin overexpres-sion group, when compared with the control group and the blank control group in Eca109 (Fig. 3A and B) and KYSE150 (Fig. 3C and D) cells. In addition, western blotting analysis indicated that the protein levels of survivin, and phosphorylation of NF-κB p65 and IKKβ were also increased in the survivin overexpression group in the Eca109 (Fig. 3B) and KYSE150 (Fig. 3D) cells. Furthermore, KYSE150 cells demonstrated increased expression levels of IKKα protein (Fig. 3D).

To further determine whether survivin recognized and bound to the IKKβ promoter in vivo, and increased its transcriptional activity, the ChIP assay was performed in Eca109 cells. DNA was immunoprecipitated using anti-survivin polyclonal antibodies and subjected to PCR with promoter-specific primers for IKKβ. Positive amplification in Eca109 cell lines demonstrated that survivin may bind the upstream 700 bp IKKβ promoters (Fig. 4A and B).

To investigate whether overexpression of survivin affects the promoter transcriptional activities of IKKβ in Eca109 and KYSE150 cells, a Luciferase reporter gene assay was performed and overexpressed survivin was observed to significantly increase the transcriptional activity of the IKKβ promoter in Eca109 cells, but not in the KYSE150 cells (Fig. 4C).

To analyze the effect of survivin on apoptosis, flow cytometric analysis with PI and Annexin V staining was performed (Fig. 5). Results demonstrated that surviving knockdown and subsequent reduction in activation of NF-κB p65 increased apoptosis in Eca109 (Fig. 5C and D) and KYSE150 (Fig. 5G and H) cells. Conversely, survivin overexpression and concomitant activation of NF-κB p65 decreased apoptosis in Eca109 (Fig. 5A and B) and KYSE150 (Fig. 5E and F) cells.

To analyze the effect of survivin on the cell cycle, flow cytometry was performed. The results indicated that survivin knockdown increased the fraction of cells arrested in the G₂/M phase in Eca109 and KYSE150 cells (Fig. 6).