The samples for the present study were collected from 84 patients diagnosed with ESCC who underwent surgical treatment at the Department of Thoracic Surgery, The Affiliated Hospital of Yangzhou University (Yangzhou, China) between January 2015 to December 2016. The age range was 49-81 years (median, 64.46 years), and 58 male and 26 females were included. None of the patients received radiotherapy or chemotherapy prior to surgery. The ESCC specimens obtained from thora-coscopic surgery were stored at -80˚C, fixed in 10% formalin at room temperature for 24 h and embedded in paraffin. The study was reviewed and approved by the Institutional Ethics Committee of the Affiliated Hospital of Yangzhou University (Yangzhou, China). All patients signed informed consent forms.

UltraVision Quanto Detection system HRP DAB was obtained from Thermo Fisher Scientific, Inc. Roswell Park Memorial Institute-1640 (RPMI-1640) and fetal bovine serum (FBS) were purchased from Gibco; Thermo Fisher Scientific, Inc. Recombinant transforming growth factor-β1 (TGF-β1) was acquired from R&D Systems. Matrigel was obtained from BD Biosciences. Anti-DJ-1 antibody was obtained from Santa Cruz Biotechnology, Inc (catalog no. sc-55572; 1:1,000 for western blotting; 1:200 for immunohistochemistry). Anti-E-Cadherin antibody was acquired from Abcam (catalog no. ab231303; 1:1,000 for western blotting; 1:1,000 for immunohistochemistry). Antibodies against vimentin (catalog no. 5741; 1:1,000 for western blotting; 1:200 for immunohistochemistry), N-Cadherin (catalog no. 13116S; 1:1,000 for western blotting), β-actin (catalog no. 4970S; 1:1,000 for western blotting), low density lipoprotein receptor-related protein 6 (LRP6) (catalog no. 2560S; 1:1,000 for western blotting), phosphorylation-LRP6 (p-LRP6) (catalog no. 2568S; 1:1,000 for western blotting), axin1 (catalog no. 2087S; 1:1,000 for western blotting) and β-catenin (catalog no. 8480S; 1:1,000 for western blotting; 1:100 for immunohistochemistry) were purchased from Cell Signaling Technology, Inc. HRP-conjugated goat anti mouse IgG (catalog no. G1006-1; 1:2,000 for western blotting) and HRP-conjugated goat anti-rabbit IgG (catalog no. HA1001; 1:2,000 for western blotting) were from Hangzhou Hua-an Medical & Health Instruments Co., Ltd. Other chemicals of analytical grade were obtained from commercial sources.

Immunohistochemical staining of human paraffin-embedded ESCC was performed using UltraVision Quanto Detection system HRP DAB, according to the manufacturer's protocol. Briefly, slides were cut to a 5-µm thickness. Following deparaffinization and rehydration with gradient alcohol ranging between 100 and 70%, the tissues were incubated in digestive enzyme for pretreatment. Sections were washed with PBS at room temperature, and then incubated in Hydrogen Peroxide Block for 10 min and Ultra V Block for 5 min at 37˚C to reduce non-specific background staining. Following washing with PBS, the sections were incubated with primary antibodies against E-cadherin, vimentin, DJ-1 and b-catenin for 20 min at 37˚C. Following washing, the sections were then incubated with Applied HRP Polymer Quanto for 10 min at 37˚C. DAB Quanto Chromogen (30 µl) was added to 1 ml DAB Quanto Substrate, mixed by swirling and applied to tissue for 5 min, followed by washing with distilled water at room temperature. Finally, the sections were counterstained with hematoxylin for 1 min at room temperature followed by dehydration and coverslip mounting. Negative controls were treated with PBS in place of primary antibodies. The stained slides were observed with a light microscope (magnification, x100 and x200) and evaluated using Image Pro Plus (version 6.0; Media Cybernetics) as previously described (20).

The human ESCC cell line ECA-109 was purchased from the Type Culture Collection of the Chinese Academy of Sciences and cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin mixture. Cells were maintained at 37˚C in a humidified atmosphere containing 5% CO₂. Prior to experiments, ECA-109 cells in the log phase of growth were cultured in six-well plates in media containing only 1% FBS for 24 h.

ECA-109 cells (1x10⁵) were plated in six-well plates 12 h before the experiment, and RPMI-1640 containing 10 µg/l TGF-β1 was subsequently added to each well for 24 h under normal culture conditions (21). The cell morphology and distribution were captured under a light microscope (magnification, x200; Olympus Corporation). Subsequently, the protein expression levels of EMT-associated proteins E-cadherin, N-cadherin, vimentin and DJ-1 were detected by western blotting.

A lentiviral vector carrying green fluorescent protein (GFP) for DJ-1 overexpression (LV-DJ-1) was applied to mediate DJ-1 overexpression and an empty lentiviral vector was applied as the negative control (LV-con). A lentiviral vector carrying GFP and encoding short interfering RNA (siRNA) targeting DJ-1 was applied to mediate DJ-1 knockdown (LV-siRNA-DJ-1) and an empty lentiviral vector was used as the negative control (LV-siRNA-con). All lentivirus vectors were constructed by Shanghai Genechem Co., Ltd. GV358 lentiviral vector was used to upregulate the expression of DJ-1 and GV248 lentiviral vector was used to knockdown the expression of DJ-1. The negative controls were also generated using GV248 and GV358, respectively. The primer sequence of DJ-1 was as follow: Forward, 5'-GAG GAT CCC CGG GTA CCG GTC GCC ACC ATG GCT TCC AAA AGA GCT CTG G-3' and reverse, 5'-TCC TTG TAG TCC ATA CCG TCT TTA AGA ACA AGT GGA G-3'. The siRNA sequence was 5'-TTA GAG AAA CAG GCC GTT A-3'. ECA-109 cells (1.0x10⁶) were seeded in six-well plates, and lentivirus (MOI, 10) with viral-plus transduction enhancer and polybrene (Shanghai Genechem Co., Ltd.) at a final concentration of 8 µg/ml were added and incubated at 37˚C for 48 h. Subsequently, the cells were treated with 10 g/ml puromycin to eliminate the uninfected cells for 72 h. The GFP positive cells were evaluated using a fluorescence microscope (Olympus Corporation) and transfection was further confirmed by western blotting.

Total proteins of each cell group were prepared using RIPA buffer containing 1 mmol/l PMSF and a protease inhibitor cocktail (Beyotime Institute of Biotechnology). Proteins were quantified using the BCA assay kit (Beyotime Institute of Biotechnology). Total proteins (30 µg/lane) from each lysate were separated by 10% SDS-PAGE and then transferred onto PVDF membranes. Membranes were blocked in blocking buffer (5% non-fat dry milk and 1% Tween-20 in PBS) for 2 h at room temperature. The membranes were incubated with the primary antibodies against DJ-1, E-cadherin, vimentin, N-cadherin, β-actin, LRP6, p-LRP6, Axin1 and β-catenin overnight at 4˚C, and then incubated with HRP-conjugated rabbit or anti-mouse antibody at room temperature for 2 h. Immunoreactive signals were visualized using ECL detection reagent (Thermo Fisher Scientific, Inc.) with the Molecular Imager Chemi Doc XRS system (Bio-Rad Laboratories, Inc.). The bands were quantified using Quantity One analysis software version 4.62 (Bio-Rad Laboratories, Inc.). The value of each protein was normalized to the β-actin level in the same sample.

Thymidine analog 5-ethynyl-2'-de-oxyuridine (EdU) (Invitrogen; Thermo Fisher Scientific, Inc.) was used to detect cell proliferation. According to the manufacturer's protocol, 2.0x10⁴ cells/well were seeded into 96-well plates and incubated at 37˚C in a 5% CO₂ incubator for 24 h. Then, 25 µM EdU was added for 2 h at 37˚C. Following washing with PBS, cells were fixed with 4% para-formaldehyde for 30 min at room temperature and terminated with 2 mg/ml glycine. The Apollo staining reaction liquid (catalog no. C10310; Guangzhou RiboBio Co., Ltd.) was then added to the wells and incubated in dark at room temperature for 30 min. Subsequently, 100 µl 0.5% TritonX-100 was applied to reduce the dye background. DAPI was diluted and used to dye the nucleus in dark at room temperature for 30 min. The results were observed and photographed using a fluorescence inversion microscope system (magnification, x100; Olympus Corporation).

To examine clonogenic ability, 500 cells were seeded in six-well plates and cultured with complete medium for 2 weeks in a 5% CO₂ incubator at 37˚C (22). Then, the cells were fixed with 99% methanol for 10 min at room temperature, and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) for 15 min at room temperature. Clones with ≥50 cells were scored under a light microscope (Olympus Corporation) and five random views were evaluated (magnification, x100).

The 96-well plates were coated with 0.04 µg/µl Matrigel (BD Biosciences) prior to the experiments. Cells (5x10⁴) were seeded in the plates for 2 h and then washed with PBS to remove non-adherent cells. MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA) was added to each well in the dark for 4 h in a 5% CO₂ incubator at 37˚C. The optical density (OD) value was measured at 490 nm. The adhesion rate was calculated as: OD of objective cells/OD of negative control cells x100%.

A Corning Matrigel invasion chamber (Corning, Inc.) was used for the Transwell assay. In the invasion assay, Matrigel gel diluted with serum-free RPMI-1640 (1:8) was added to the upper chamber. A single cell suspension in serum-free RPMI-1640 was added to the upper chamber at a density of 2x10⁴ cells/well and incubated for 24 h (23). The non-invading cells on the upper side of the chamber were removed using cotton swabs. The membrane containing the invaded cells was fixed with 99% methanol for 10 min at room temperature and stained with 0.1% crystal violet for 10 min at room temperature. Migration assays were performed using the same procedure, except that the polycarbonate membrane was not coated with Matrigel. Each experiment was repeated three times. The number of invading and migrating cells was detected by light microscopy (magnification, x200). The invasion and migration capacity of cells was calculated as follows: Cell number of the treatment group/cell number of the control group x100%.

To evaluate the exact mechanism, the present study investigated the effect of β-catenin inhibition on the role of DJ-1 in ESCC malignant behaviors using XAV939 (MedChemExpress), a small molecule inhibitor of Wnt/β-catenin. LV-DJ-1 cells were cultured in 6-well plates in advance, then 10 mM XAV939 was added for 24 h in a 5% CO₂ incubator at 37˚C and the same concentration of DMSO was used as a negative control. The treated cells were then subjected to a cell proliferation assay, cell clone assay, cell adhesion assay, Transwell assay and western blotting.

A total of 20 4-week-old female athymic nude BALB/c mice were obtained from the Comparative Medicine Laboratory Animal Center of Yangzhou University (Jiangsu, China). All mice were raised in a specific pathogen-free atmosphere environment at 25˚C with a 12 h light/dark cycle and allowed free access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee of Yangzhou University and performed in accordance with internationally accepted guidelines on the use of laboratory animals. Nude mice were randomly assigned to four groups; each group contained 5 mice. ECA-109/LV-con, ECA-109/LV-DJ-1, ECA-109/LV-siRNA-DJ-1 and ECA-109/LV-siRNA-con cells (1x10⁶) were separately inoculated into the peritoneal cavity of nude mice (24). Because the intraperitoneal nodules could not be measured directly for tumor progression, we referred to relevant literature and chose 28 days as the total time of the experiment (24). In the 28 days, tumor progression was assessed by abdominal palpation and in vivo imaging of the small animals using IVIS^® Lumina Series III (PerkinElmer, Inc.) every 2 days. Isoflurane (3%) was used for induction and 1.5% isoflurane was used for maintenance of anesthesia in the process of imaging small animals in vivo. However, due to the poor imaging effect of GFP in cells and the high background fluorescence in the picture, tumor progress was roughly evaluated according to the intensity of the strongest fluorescence. During the process, all the mice were alive, and almost all of them showed wasting, but they were able to eat and drink normally; therefore, the experiment was continued until the end of the experiment period. A total of 28 days following injection, all the mice were anesthetized with 3% isoflurane gas inhalation and sacrificed by cervical dislocation, then a laparotomy was performed. Peritoneal dissemination and organ metastasis were examined. The peritoneal nodules and liver were fixed in 10% formalin and embedded in paraffin for pathological analysis.

All the experiments were repeated three times. Protein expression levels and clinicopathological features were compared using the χ² test. Correlations between DJ-1, E-cadherin and vimentin in clinical samples were analyzed using Pearson's correlation analysis. Other data are presented as the mean ± standard deviation, and groups were compared using Student's t-test. One-way ANOVA followed by Fisher's least significant difference post hoc test was used for multiple comparisons. All statistical analysis was performed using SPSS version 16.0 software (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.

In order to elucidate the association between DJ-1 and EMT in ESCC, immunohistochemistry analyses were first performed to detect the expression levels of DJ-1, vimentin and E-cadherin in 84 human ESCC tissue specimens (Fig. 1A). The associations between the clinical pathological features of patients with ESCC and the DJ-1, E-cadherin ad vimentin immunohis-tochemical staining scores are summarized in Table I. The data demonstrated that high expression of DJ-1 was significantly associated with the lymph node metastasis and distant metastasis. In addition, high expression of vimentin and low expression of E-cadherin were significantly associated with the tumor cell differentiation, pT status, lymph node metastasis and distant metastasis (P<0.05).

Furthermore, the expression levels of EMT biomarkers E-cadherin and vimentin were significantly correlated with DJ-1. As presented in Fig. 1B and C, DJ-1 expression was positively correlated with vimentin expression (r=0.739, P<0.001) and negatively correlated with E-cadherin (r=-0.770, P<0.001). These results collectively suggest that DJ-1 may be closely associated with metastasis and the EMT in ESCC.

To study the DJ-1 expression in EMT progress, EMT was induced in ECA-109 cells using 10 ng/ml TGF-β1. Following 24 h, cells were photographed under a light microscope and examined by western blotting. As presented in Fig. 2A, the cell morphology of the TGF-β1 group exhibited spindle-like shapes. The distribution of cells in the TGF-β1 group was more extensive and changes in migration patterns were observed compared with the control group. Furthermore, the expression levels of EMT biomarkers were significantly different following TGF-β1 treatment; E-cadherin was significantly decreased, and N-cadherin and vimentin were significantly increased (P<0.01). In addition, DJ-1 expression was significantly increased following TGF-β1 treatment (P<0.01; Fig. 2B). Thus, DJ-1 was highly expressed during EMT in ECA-109 cells.

To validate the role of DJ-1 in tumor metastasis and EMT, lentivirus vectors were used to overexpress DJ-1 in Eca-109 cells. Successful overexpression of DJ-1 of was verified by fluorescence observation of the GFP signal (Fig. 3A). In addition, western blotting demonstrated that DJ-1 expression was significantly increased in LV-DJ-1 cells compared with control cells (P<0.01; Fig. 3B). As presented in Fig. 3C, cell proliferation was higher following DJ-1 overexpression compared with the control group, as the Edu staining was stronger. In order to further observe the change of cell proliferation ability, a cell clone assay was performed. This revealed that LV-DJ-1 cells had a larger colony size and significantly more colonies (189.33±15.50) compared with the control cells (106±12.77) (P<0.01; Fig. 3D).

A Transwell assay was performed to detect the migration and invasion ability of DJ-1 overexpressed cells. As presented in Fig. 4A, LV-DJ-1 cells had a significantly higher migration and invasion ability compared with the LV-con group (P<0.01). Similarly, the adhesion rate was significantly higher in LV-DJ-1 cells compared with the LV-con cells (P<0.05; Fig. 4B). Western blotting results demonstrated that DJ-1 overexpression could significantly reduce the E-cadherin expression, and significantly increase the vimentin and N-cadherin expression (P<0.01; Fig. 4C), which indicates DJ-1 promotes the EMT process. LRP6, p-LRP6, axin1 and β-catenin are key proteins of the canonical Wnt/β-catenin signal pathway (13). It was identified that in LV-DJ-1 cells, LRP6, p-LRP6 and β-catenin expression levels were significantly upregulated, while axin1 expression was significantly downregulated compared with control cells (P<0.01; Fig. 4D). However, the ratio of p-LRP6 to total LRP6 protein had no significant change between the control group and DJ-1 overexpressed group (P>0.05; Fig. 4D). Therefore, this suggests that a high expression of DJ-1 does not effect the phosphorylation of LPR6.

To further confirm the effects of DJ-1 in tumor metastasis and EMT of ESCC, lentivirus vectors encoding siRNAs targeting DJ-1 were used to knockdown DJ-1 expression in ECA-109 cells. As presented in Fig. 5A, DJ-1 was knocked-down, according to the extent of green fluorescence. Additionally, western blot assay revealed that the protein expression of DJ-1 was significantly reduced in LV-siRNA-con cells (P<0.01; Fig. 5B). These results suggest that the DJ-1 low-expression cell model was successfully constructed. Edu staining assay demonstrated that LV-siRNA-DJ-1 cells had a low proliferative capacity compared with control cells, as the red fluorescence was enhanced. (Fig. 5C). Furthermore, the ability of cells in the LV-siRNA-DJ-1 group to proliferate and form clones was significantly lower compared with the control group (P<0.01; Fig. 5D).

In contrast to DJ-1 overexpressed cells, the LV-siRNA-DJ-1 cells had significantly lower migration, invasion and adhesion abilities compared with LV-siRNA-control cells (P<0.01; Fig. 6A and B). The E-cadherin expression was significantly higher, while N-cadherin and Vimentin expression were significantly lower in LV-siRNA-DJ-1 cells compared with control cells (P<0.01; Fig. 6C). Furthermore, LRP6, p-LRP6 and b-catenin expression were significantly downregulated, while Axin1 expression was significantly upregulated in LV-siRNA-DJ-1 cells (P<0.01; Fig. 6D). Finally, the ratio of phosphorylated LRP6 to total LRP6 protein was significantly increased in LV-siRNA-DJ-1 cells compared with control cells.

In order to investigate the exact mechanism of the Wnt/β-catenin signaling pathway in the regulation of DJ-1 in tumor malignant behaviors, the β-catenin inhibitor XAV939 was used in in vitro studies. LV-DJ-1 cells were treated with XAV939; as presented in Fig. 7A, the proliferation of LV-DJ-1 cells treated with XAV939 was decreased compared with LV-DJ-1 cells. In the colony formation assay, cells treated with XAV939 had a significantly smaller number of colonies compared with the control LV-DJ-1 cells (P<0.01; Fig. 7B). In the Transwell assay, LV-DJ-1 cells treated with XAV939 demonstrated significantly reduced abilities to migrate and invade compared with LV-con group (P<0.01; Fig. 7C). Similarly, the adhesion ability was significantly decreased in the XAV939 group compared with the LV-con group (P<0.01; Fig. 7D). Western blotting results demonstrated that XAV939 treatment could signifi-cantly increase the E-cadherin, while it significantly reduced the vimentin and N-cadherin expression levels compared with the untreated LV-DJ-1 cells (P<0.01; Fig. 7E), which indicates XAV939 inhibited the EMT process promoted by DJ-1. Additionally, LRP6, p-LRP6 and b-catenin expression levels were significantly downregulated, while Axin1 expression was significantly upregulated compared with control cells, and the ratio of phosphorylated LRP6 to total LRP6 protein was significantly increased in XAV939-treated cells compared with the control untreated LV-DJ-1 cells (P<0.05; Fig. 7F), These data suggest XAV939 could reverse tumor malignant behavior induced by overexpression of DJ-1.

Since the important roles of DJ-1 in metastasis and EMT have been demonstrated by clinical analysis and in vitro experiments, its biological effect on ESCC metastasis and EMT was further examined in vivo using a nude mice abdominal transplantation model. In vivo imaging of the small animals was also used to observe tumor growth dynamically. However, due to the poor imaging effect of green fluorescent protein in cells and the high background fluorescence in the picture, tumor progress was roughly estimated according to the intensity of the strongest fluorescence. In the late stage of tumor growth, differences were observed between each group. At day 28, the LV-DJ-1 group had a larger area and stronger fluorescence compared with the control, while the LV-siRNA-DJ-1 group had a smaller area of fluorescence (Fig S1). The numbers of peritoneal dissemination nodules were examined and the liver metastases were removed and analyzed. As presented in Fig. 8A and B, the number of peritoneal dissemination nodules in LV-DJ-1 group was significantly increased compared with the LV-con group, while in the LV-siRNA-DJ-1 group, the number of peritoneal dissemination nodules was significantly reduced compared with the LV-siRNA-con group (P<0.01). Notably, liver metastases in the LV-DJ-1 group exhibited a larger volume and contained a larger number of nodules (Fig. 8C). By contrast, in the LV-siRNA-DJ-1 group, liver metastases had a smaller volume and fewer nodules (Fig. 8D). Immunohistochemistry results further explained the mechanism of DJ-1 promoting tumor metastasis. Immunohistochemical staining (Fig. 8E) revealed increased DJ-1, vimentin and β-catenin levels, and decreased E-cadherin levels in the LV-DJ-1 group compared with the control group. In addition, staining demonstrated decreased DJ-1, vimentin and β-catenin levels, and increased E-cadherin levels in the LV-siRNA-DJ-1 group compared with the control group.