ESCC and adjacent non-tumor tissues (at least 3 cm from the primary tumor) were collected from 56 patients with ESCC who underwent surgical resection from September 2011 to April 2013. Patients with ESCC who received preoperative chemo- or radiotherapy were excluded from the study. All of the tissues were stored at −80°C until further use. The present study was approved by the Ethics Committee of the First People's Hospital of Chenzhou City (Chenzhou, China). Written informed consent was provided by all the participants prior to the study. The follow-up time was 5 years from surgical resection.

The human Het-1A esophageal epithelial cell line, and four human ESCC cell lines, TE-9, ECA109, KYSE150 and EC9706, were obtained from the Cell Bank of the Chinese Academy of Sciences. The cell lines were cultured in DMEM with 10% FBS (both from Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO₂. For cell transfection, the TE-9 and EC9706 cells were transfected with 100 nM CCEPR short hairpin (sh)RNA1 (cat. no. n352462), CCEPR shRNA2 (cat. no. n352463), or negative control (NC) shRNA (cat. no. 4457289) using Lipofectamine^® 2000 (all from Thermo Fisher Scientific, Inc.). Reverse transcription-quantitative PCR (RT-qPCR) was performed to examine the mRNA expression level of CCEPR, 48 h following transfection.

TRIzol^® reagent (Thermo Fisher Scientific, Inc.) was used to extract total RNA from tissues and cell lines. The total RNA was then reversed transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.). The mRNA expression levels were then determined using a SYBR^® Green Real-time PCR Master Mix (Toyobo Life Science) on a Roche 480 Real-Time PCR system (Roche Diagnostics). The following thermocycling conditions were used: Initial denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. The relative expression was determined with the 2^−ΔΔCq method (21). The primer sequences used were as follows: CCEPR forward, 5′-AAGGTCCCAGGATACTCGC-3′ and reverse, 5′-GTGTCGTGGACTGGCAAAAT-3′; GAPDH forward, 5′-CTGGGCTACACTGAGCACC-3′ and reverse, 5′-AAGTGGTCGTTGAGGGCAATG-3′.

Transfected TE-9 and EC9706 cells (5,000 cells/well) were seeded in 96-well plates, then cultured for 0, 24, 48 and 72 h. Next, 10 µl CCK-8 solution (Thermo Fisher Scientific, Inc.) was added to the cells and incubated at 37°C for 2 h, according to the manufacturer's instructions. The absorbance (450 nm) was determined using a microplate reader (Bio-Rad Laboratories, Inc.).

Transfected TE-9 and EC9706 cells (300 cells/well in 6-well plates) were cultured in DMEM supplemented with 10% FBS for 2 weeks. Then, the cells were stained with a 0.5% crystal violet solution at room temperature for 5 min. The colony numbers (containing >50 cells) were counted.

Transfected TE-9 and EC9706 cells were washed with Dulbecco's phosphate (DPBS; Thermo Fisher Scientific, Inc.) and fixed in 75% ethanol at 4°C for 3 h. After washing with PBS, twice, the cells were stained with both FITC Annexin V and propidium iodide (both from BD Biosciences) at room temperature for 30 min. The cell apoptosis rate was examined using a FACScan flow cytometer and analyzed using the Accuri C6 system software v1.0 (both from BD Biosciences).

For the cell migration assay, transfected TE-9 and EC9706 cells in 12-well plates (5×10⁵ cells/well) were cultured at 37°C, to ~95% confluence. A 100 µ sterile pipette tip was used to scratch the cells to generate a wound. The cells were washed with DPBS and were added to each well with serum-free DMEM. At this time point (indicated as 0 h), images of the wound were obtained, using a light microscope (×40). Then, the cells were incubated at 37°C for 24 h, and images of the wound were obtained again, under a light microscope (×40). Wound closure rate is calculated based on wound area relative to the original size using ImageJ (version 1.8; National Institutes of Health).

Matrigel-coated Transwell chambers (BD Biosciences, USA) were used to perform the cell invasion assay. The precoating was performed at room temperature for 30 min. In brief, transfected TE-9 and EC9706 cells (50,000 cells/well) in 400 µl serum-free DMEM were added to the upper chamber. Next, 300 µl DMEM with 10% FBS was added to the lower chamber. After incubation at 37°C for 24 h, the invading cells were stained with a 0.1% crystal violet solution at room temperature for 10 min. Then, images were obtained using a light microscope (×200).

Total protein was extracted from the tissues and cells using a RIPA buffer (Thermo Fisher Scientific, Inc.). The protein concentrations were determined with a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). The proteins were separated using 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.). After blocking with 5% skimmed milk overnight at 4°C, the membranes were incubated with the primary antibodies against caspase-3 (cat. no. ab13847; 1:500), Bcl2 (cat. no. ab32124; 1:200), Bax (cat. no. ab32503; 1:500), matrix metalloproteinase [(MMP)2; cat. no. ab97779; 1:200], MMP9 (cat. no. ab38898; 1:500), N-cadherin (cat. no. ab76057; 1:500), vimentin (cat. no. ab45939; 1:500), E-cadherin (cat. no. ab227639; 1:200), and GAPDH (cat. no. ab9485; 1:500) at room temperature for 5 h and then with the HRP-conjugated goat anti-rabbit secondary antibody (cat. no. ab6721; 1:10,000) at room temperature for 2 h (all antibodies were obtained from Abcam). A western lightning™ chemiluminescence reagent plus kit (Thermo Fisher Scientific, Inc.) was used to visualize the bands, while the ImageJ software v1.48 (National Institutes of Health) was used to determine the protein expression level.

The data are expressed as the mean ± SD and the experiments were repeated 3 times. SPSS v19.0 (IBM Corp.) was used for statistical analysis. A one-way ANOVA followed by Tukey's post hoc test was used to analyze the differences between more than two groups, while an unpaired Student's t-test was used to analyze the differences between 2 groups. Based on the median expression value (2.372) of CCEPR, the patients were divided into a high and a low CCEPR expression level group. The associations between CCEPR mRNA expression levels and the clinicopathological characteristics of patients with ESCC were analyzed using a χ² test. The survival analysis was performed using Kaplan-Meier analysis and log-rank test. P<0.05 was considered to indicate a statistically significant difference.

In the present study, the mRNA expression levels of CCEPR in ESCC tissues was determined using RT-qPCR. As shown in Fig. 1A, the expression levels of CCEPR were significantly higher in ESCC tissues compared with that in adjacent non-tumor tissues. In addition, the CCEPR mRNA expression level was also significantly higher in ESCC cell lines compared with that in the Het-1A cell line (Fig. 1B).

We hypothesized that the increased mRNA expression of CCEPR might be involved in the development and progression of ESCC. To investigate this hypothesis, the clinical significance of CCEPR mRNA expression in ESCC was determined. Based on the median expression value (2.372) of CCEPR, the patients were divided into a high and a low CCEPR expression level groups. As shown in Table I, a χ² test data indicated that high CCEPR expression was significantly associated with advanced TNM stage, as well as lymph node metastasis, suggesting that overexpression of CCEPR may play an important role in ESCC progression. Subsequently, the overall survival time was significantly shorter for patients with ESCC and high CCEPR mRNA expression levels compared with that in patients with low CCEPR expression levels (Fig. 1C). Thus, upregulation of CCEPR may predict poor prognosis in ESCC.

Then, the function of CCEPR in regulating ESCC cells in vitro was investigated. As CCEPR was significantly upregulated in the ESCC cell lines, and the TE-9 and EC9706 cell lines showed the highest mRNA expression levels of CCEPR, the TE-9 and EC9706 cell lines were transfected with two shRNAs to downregulate CCEPR expression. CCEPR was downregulated in the CCEPR shRNA 1 and shRNA 2 groups compared with that in the NC shRNA group, in both cell lines (Fig. 2A and B). A CCK-8 assay was then performed to determine the effects of CCEPR downregulation on ESCC cell proliferation and the results showed that the number of TE-9 and EC9706 cells was significantly lower in the CCEPR shRNA groups compared with that in the NC shRNA group (Fig. 2C and D). Thus, CCEPR may promote ESCC cell proliferation.

The reduced cell proliferation caused by CCEPR downregulation may be due to increased cell apoptosis. Thus, flow cytometry was performed to examine the rate of cell apoptosis in each experimental group. As shown in Fig. 3A and B, apoptosis of both the ESCC cell lines transfected with 2 types of shRNA was significantly increased following silencing of CCEPR mRNA expression compared with that in the NC group. Subsequently, the protein expression levels of several key apoptotic-related genes, including pro-apoptotic Bax and caspase-3 and anti-apoptotic Bcl2 were investigated. Knockdown of CCEPR significantly increased the protein expression levels of Bax and caspase-3, while the protein expression levels of Bcl2 was decreased in both ESCC cell lines transfected with shRNA (Fig. 3C and D).

The effects of CCEPR knockdown on the migratory and invasive abilities of ESCC cells was subsequently investigated. The results from the wound healing assay indicated that the migration of TE-9 and EC9706 cells was significantly inhibited following downregulation of CCEPR (Fig. 4A and B). Similarly, silencing CCEPR expression also downregulated the invasive abilities of both ESCC cell lines (Fig. 4C and D). The protein expression levels of MMP2 and MMP9 were then examined using western blot analysis. As shown in Fig. 5A and B, downregulation of CCEPR significantly decreased the protein expression levels of MMP2 and MMP9. Then, the effects of CCEPR downregulation on EMT in both the ESCC cell lines was investigated and the results showed that silencing CCEPR increased the protein expression levels of E-cadherin, while the protein expression levels of N-cadherin and vimentin were decreased, indicating that EMT was reduced (Fig. 5C and D). Therefore, CCEPR may promote EMT in ESCC cell lines.