A total of 110 patients with complete clinical records who were treated at the Department of Thoracic Surgery, Provincial Hospital Affiliated to Shandong University (Jinan, China) between January 2011 and January 2013 were enrolled for this retrospective study. The patients included 89 males and 21 females, with a mean age of 61 years (range, 31-79 years). The clinical information of all patients is presented in Table I. The inclusion criteria were as follows: i) Patients subjected to Ivor-Lewis esophagogastrectomy with thoracic and abdominal two-field lymph node dissection (14) to achieve complete tumor resection as per the 2009 Union for International Cancer Control (UICC) standard for midthoracic ESCC; ii) patients restaged according to the 2009 UICC tumor-node-metastasis (TNM) staging guidelines for EC; iii) no residual cancer cells under the upper and lower cutting edge, and a lateral margin without residual focus, as demonstrated by pathological examination; iv) >12 lymph node dissections; v) no preoperative chemotherapy or radiotherapy; vi) no severe preoperative complications; and vii) at least a complete 3-year follow-up review and first specific recurrence location recorded. ESCC tissue and corresponding healthy esophageal mucosa (CHEM) tissue samples were collected from each patient. The CHEM tissue samples were obtained 5 cm from the edge of the ESCC tissues, and they were examined by light microscopy to confirm the absence of necrosis, deterioration or tumor.

IHC analysis was conducted as described previously (15). Briefly, ESCC and CHEM tissues were fixed with 4% paraformaldehyde at 4°C for 24 h, paraffin-embedded and cut into 5-μm sections. Following blocking with goat serum for 30 min at 25°C for 40 min, sections were incubated with anti-SKA1 primary antibody (1:50; cat. no. ab91550; Abcam) at 37°C for 1 h. After three washes with PBS, the sections incubated with a biotin goat anti-rabbit IgG H&L secondary antibody (1:2,000; cat. no. ab6720; Abcam) for 30 min at 37°C, followed by incubation with streptavidin-horseradish peroxidase for 5 min at 37°C. Subsequently, 3,3'-diaminobenzidine was added for visualization after washing with PBS three times. Finally, the sections were counterstained with hematoxylin at 25°C for 1 min, air-dried and mounted on a cover slip. SKA1 expression was observed under a light microscope at three magnifications (x100, x200 and x400).

The immunohistochemical score (IHS) consisted of the proportion score (percentage of positively stained cells) and the staining intensity score. The proportion score was described as follows: 0 (<5%), 1 (5-24%), 2 (25-49%), 3 (50-74%) and 4 (75-100%). The staining intensity score was calculated as follows: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The proportion score was multiplied by the staining intensity score to obtain an IHS for each section. SKA1 expression was rated as follows: High expression, HIS ≥4; and low expression, HIS <4. Two experienced pathologists blinded to the patient data independently scored the samples and agreed on a result through reanalysis and discussion.

All patients were regularly followed-up every 3-6 months after surgery for a general physical examination, abdominal ultrasound, chest and upper abdominal contrast-enhanced computed tomography scan, and positron emission tomography. Biopsies were used based on specific imaging and clinical examinations. The follow-up was completed in January 2017 with a maximum follow-up period of 60 months.

The human ESCC cell lines TE-1, EC9706 and Eca-109, a human esophageal epithelial cell line HET-1A and 293T cells were purchased from the Cell Bank of Chinese Academy of Sciences. ESCC cell lines and HET-1A were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.), and 293T cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.), which were both supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin G and 100 μg/ml streptomycin (Sigma-Aldrich; Merck KGaA). All cell lines were incubated at 37°C in 5% CO₂.

A short hairpin RNA (shRNA) targeting human SKA1 (SKA1-shRNA; 5'-GGA GAT GAG ATC ATT GTA A-3') was inserted into the lentivirus expression plasmid pFH-L (Genechem, Inc.). Non-silencing shRNA (5'-GGA GAT GAG ATC ATT GTA A-3') was used as a control (control-shRNA). Recombinant pFH-L plasmids (20 μg) and two other helper plasmids [pVSVG-I (15 μg) and pCMV-R8.92 (10 μg)] (Shanghai Hollybio) were transfected into 293T cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) to package lenti-virus expressing SKA1-shRNA or control-shRNA. After 48 h, the supernatant was collected and the concentrated supernatant was used to infect TE-1 cells (multiplicity of infection, 10) following titer determination. TE-1 cells infected with lentivirus expressing SKA1-shRNA or control-shRNA were termed shSKA1 or shCtrl, respectively.

TE-1 cells of the shSKA1 or shCtrl groups were cultured for 48 h and cells were then collected for RNA extraction. TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) was used according to the manufacturer's protocol to extract total RNA from tumor tissues and cultured cells. Complementary DNA (cDNA) was prepared using superscript III reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. qPCR was performed using SYBR Green qPCR SuperMix (Invitrogen; Thermo Fisher Scientific, Inc.) to detect mRNA expression with the following conditions: 95°C for 2 min; 40 cycles of 95°C for 15 sec and 60°C for 1 min. GAPDH cDNA was amplified and used as an internal standard. The primers used were as follows: SKA1 forward, 5'-TTC CCA TTT GCC TCA AGT AAC AG-3' and reverse, 5'-GGA ACA CCA TTG AAC TCA TCA CAA G-3'; and GAPDH forward, 5'-GCA CCG TCA AGG CTG AGA AC-3' and reverse, 5'-TGG TGA AGA CGC CAG TGG A-3'. Relative expression was calculated using the 2^-ΔΔCq method (16).

TE-1 cells of the shSKA1 or shCtrl groups were cultured for 48 h and cells were then collected for total protein extraction. Total protein was extracted using ice-cold radioimmunoprecipitation assay extraction and lysis buffer (Takara Bio, Inc.) containing a proteinase inhibitor cocktail (Takara Bio, Inc.). The concentration of total protein was determined using a BCA Protein assay kit (Nanjing KeyGen Biotech Co., Ltd.), according to the manufacturer's protocol. The extracted proteins (30 μg) were separated by 10% SDS-PAGE and the separated protein bands were transferred onto nitrocellulose membranes. The membranes were incubated with rabbit anti-SKA1 antibody (1:500; cat. no. ab91550; Abcam) and rabbit anti-GAPDH antibody (1:10,000; cat. no. ab181602; Abcam) at 4°C overnight, followed by treatment with horseradish peroxidase-conjugated goat anti-rabbit IgG H&L secondary antibody (1:10,000; SouthernBiotech) for 40 min at 25°C. The protein bands were visualized using enhanced chemiluminescence substrate (Thermo Scientific Inc.). The expression of all proteins was normalized against that of GAPDH.

TE-1 cells of the shSKA1 and shCtrl groups were seeded into 96-well plates (1,500 cells/well) and incubated at 37°C under 5% CO₂ for 4 days. The cell count assay was performed using a Celigo Imaging Cytometer (Nexcelom Bioscience), according to the manufacturer's protocol. The captured cell images were analyzed using Celigo software (Nexcelom Bioscience).

CellTiter 96^® AQueous One Solution assay kit was used to detect cell viability. Briefly, TE-1 cells (2x103/well) of the shSKA1 and shCtrl groups were seeded in 96-well plates. After 1, 2 or 3 days of incubation, the cells were treated with 5 mg/ml MTS reagent and incubated for 4 h at 37°C. Following incubation, the supernatant was used to measure the optical density (OD) at a wavelength of 490 nm using a microplate reader. All experiments were performed in triplicate. Cell viability was calculated using the following equation: Cell viability (%)=(OD value of tested group/OD value of shCtrl group) x100%.

TE-1 cells of the shSKA1 and shCtrl groups were seeded in six-well plates (4x105/well). After 2 days, the cell cycle was analyzed using propidium iodide (Sigma-Aldrich; Merck KGaA) staining for 30 min at 37°C and flow cytometry (Cytomics FC 500 MPL, Beckman Coulter, Inc.). Results were analyzed using ModfitLT, version 2.0 (Verity Software House Inc.). All experiments were performed in triplicate.

The ApoScreen Annexin V Apoptosis kit (Southern Biotech) was used to label apoptotic cells, according to the manufacturer's protocol. Following culture for 48 h, TE-1 cells of the shSKA1 or shCtrl groups were harvested and stained with Annexin V-APC, followed by flow cytometry analysis (FACSCalibur; Becton, Dickinson and Company) after 15 min of incubation at 25°C. Results were analyzed using BD FACSDiva software (version 8.0; Becton, Dickinson and Company). All experiments were performed in triplicate.

TE-1 cells (4x104) of the shSKA1 and shCtrl groups were seeded onto the upper chamber of a Transwell plate in serum-free RPMI-1640 medium. RPMI-1640 medium (600 μl) with 20% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) was added to the lower chamber of the Transwell plate. After 48 h, the migration of cells to the lower chamber of the Transwell plate was determined. Cells were stained with 0.1% crystal violet in 20% ethanol for 10 min at 25°C, and the number of cells was counted in five independent fields under a light microscope (magnification, x200). All experiments were performed in triplicate.

SPSS 19.0 software (SPSS, Inc.) was used for all statistical analyses. The significant difference in SKA1 expression between tumor and paratumor samples was evaluated using a χ2 test. The Kaplan-Meier method was used to analyze the survival rate and the significance of the survival difference was calculated using a log-rank test. Independent prognostic factors were determined through Cox regression multivariate analysis. Statistical comparisons between two groups were analyzed using an unpaired Student's t-test. Statistical analysis for more than two groups was performed using one-way ANOVA followed by a post-hoc least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

As presented in Fig. 1A, SKA1 expression was predominantly detected in the cytoplasm of tumor cells. SKA1 protein expression was markedly higher in ESCC tissues compared with in CHEM tissues (Fig. 1A). Of the 110 samples, 68 (61.8%) tumor samples and only 29 (26.4%) CHEM samples exhibited a high SKA1 expression. In addition, SKA1 mRNA expression was significantly higher in ESCC tissues compared with in CHEM tissues (Fig. 1B).

The association between SKA1 expression and clinicopathological parameters is presented in Table I. No significant associations were identified between SKA1 expression and age, sex, tumor size or pathological T stage. However, SKA1 overexpression was significantly associated with differentiation, pathological N stage and pathological TNM stage (Table I).

Follow-up data were available for all patients included in the present study. The results of Kaplan-Meier analysis indicated that the overall 5-year survival rate was 27.27%. Patients with high SKA1 protein expression had a 5-year survival rate of 17.64%, compared with a survival rate of 42.85% for patients with low SKA1 expression (P<0.01; Fig. 2). The results of multivariate analysis revealed that pathological TNM stage and SKA1 protein overexpression were independent prognostic factors for ESCC (Table II).

The mRNA expression of SKA1 in the human ESCC cell lines TE-1, EC9706 and Eca-109, and a human esophageal epithelial cell line HET-1A was investigated through RT-qPCR analysis. It was identified that the expression of SKA1 mRNA was significantly higher in TE-1, EC9706 and Eca-109 cells compared with in HET-1A cells (Fig. 3). Among the ESCC cell lines, the SKA1 mRNA level was highest in TE-1 cells. Therefore, TE-1 cells were selected for the following assays.

To evaluate the functions of SKA1 in ESCC cells, SKA1 expression was knocked-down by lentivirus-mediated shRNA transfection. The mRNA and protein expression levels of SKA1 were evaluated using RT-qPCR and western blot analysis, respectively. In comparison with the shCtrl group, the cells transfected with shSKA1 demonstrated a marked reduction in the mRNA and protein expression levels of SKA1 (Fig. 4). Therefore, lentivirus-mediated shRNA knockdown was demonstrated to serve as an effective strategy to knockdown SKA1 expression in EC cells.

Celigo image cytometry analysis and an MTS assay were used to evaluate the effect of SKA1-knockdown on the proliferation ability of TE-1 cells. As presented in Fig. 5A and B, the cell number in the shSKA1 group was significantly lower compared with that in the shCtrl group at days 1-4. In addition, the results of the MTS assays demonstrated that the percentage of cell viability in the shSKA1 group was significantly lower compared with that of the shCtrl group at days 1, 2 and 3 (Fig. 5).

It was identified that the proportion of cells in the G1 and S phases was significantly higher in the shSKA1 group compared with in the shCtrl group (Fig. 6A). In addition, apoptosis analysis revealed that the apoptosis rate was significantly higher in the shSKA1 group compared with in the shCtrl group (Fig. 6B). In summary, these results suggest that silencing SKA1 expression in TE-1 cells may lead to cell cycle arrest at the G1/S phase and promote apoptosis.

To determine the role of SKA1 expression in ESCC metastasis, the present study investigated the association between SKA1 expression and TE-1 cell migration ability. The results demonstrated that the number of migratory cells per field was significantly lower in the shSKA1 group compared with in the shCtrl group, indicating that the migration ability was significantly reduced in the cells lacking SKA1 expression compared with the control cells (Fig. 7).