Human ESCC and adjacent tissues used in the present study were obtained from the Nanjing Medical University affiliated Suzhou hospital (Jiangsu, China). These tissues were resected from patients before chemotherapy and radiation therapy, and were immediately frozen and stored at −80°C for reverse transcriptase (RT)-PCR and real-time PCR analysis. All patients gave signed, informed consent for their tissues to be used for scientific research. The study was approved by the Institutional Ethics Committee of Nanjing Medical University Affiliated Suzhou Hospital.

Human esophageal cancer cell lines ECA-109 and TE-1 were obtained from the Shanghai Cell Bank (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from HyClone) in a humidified atmosphere, with 5% CO₂ at 37°C.

The shRNA construct against REV3L (shREV3L) and the control plasmid (shNC) were purchased from Shanghai GenePharma (Shanghai, China). The shREV3L construct or control vector was transfected into ECA-109 and TE-1 cells using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocols. Stable clones were selected in medium containing G418 (500 µg/ml) (Sigma-Aldrich, St. Louis, MO, USA) for 4 weeks. Individual clones were isolated and expanded for further characterization.

Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies) following the manufacturer's instructions. For reverse transcription, 1.0 µg of RNA/sample was reverse transcribed using an oligo(dT)₁₂ primer and SuperScript II reverse transcriptase (Invitrogen Life Technologies). The primers for REV3L and β-actin were as follows: forward, 5′-CGCGTCAGTTGGGACTTAAG-3′ and reverse, 5′-ACTATCGCCAACCTCAATGC-3′ (REV3L); forward, 5′-AGCGAGCATCCCCCAAAGTT-3′ and reverse, 5′-GGGCACGAAGGCTCATCATT-3′ (β-actin). RT-PCR was conducted using the 2X Taq PCR Master Mix (Takara Bio, Dalian, China) according to the manufacturer's instructions. The PCR products were separated by electrophoresis on 1.5% agarose gels, and the quantification of each band was performed using Quantity One software (Bio-Rad).

Cells were fixed with 4% paraformaldehyde, washed with phosphate-buffered saline (PBS) and permeabilized with 1% Triton X-100 in PBS. Samples were then blocked with blocking solution (PBS containing 10% BSA and 1% triton X-100) and incubated overnight at 4°C with the REV3L antibody (1:1,000; Abnova, Taiwan, China). After washing, the Rhodamine-labeled goat anti-rabbit antibody (1:100; KPL, Gaithersburg, MD, USA) was added and incubated for 1 h at room temperature. Nuclear counterstaining was performed using 4′6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Cells were visualized under a fluorescence microscope (Eclipse 80i; Nikon, Tokyo, Japan).

Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) (Beyotime Biotechnology, China). A total of 2×10³ cells/well were seeded in triplicate into 96-well plates for 1–6 days. Then, 10 µl of CCK-8 solution was added to each well, and incubated for 2 h at 37°C. The optical density (OD) was measured at 450 nm with a microplate reader (Thermo, Waltham, MA, USA). The viability index was calculated as the experimental OD value/the control OD value. For 5-fluorouracil (5-FU) treatment experiments, cells (5×10³) were initially plated in quadruplicate into 96-well plates. Twenty-four hours later, the cells were treated with various concentrations of 5-FU for 48 h. Cell viability was measured as described above. Three independent experiments were performed.

The invasive potential of cells was evaluated using 24-well Transwell inserts (Costar, New York, NY, USA). The inserts were pre-coated with 40 µl Matrigel (1:4 dilution; BD Biosciences, San Jose, CA, USA). Then, 1×10⁵ cells (200 µl) in serum-free medium were added to the upper chambers. The lower chambers were filled with medium that contained 10% FBS. After incubation for 24 h, the inserts were fixed with 3.7% paraformaldehyde/PBS and stained with 2% crystal violet. The cells remaining in the upper chambers were scraped off, and the invading cells in the lower chambers were photographed under a microscope.

A total of 5×10⁵ cells/well were seeded in triplicate into 6-well plates. Twenty-four hours later, the cells were treated with 50 and 100 µM 5-FU for 24 or 48 h. Cell cycle analysis was performed using the propidium iodide (PI) single staining method. Cells were collected and fixed with 70% ice-cold ethanol at 4°C overnight. Then, the cells were washed with PBS and stained with PI (100 µg/ml) for 30 min in the dark before analysis. The cell cycle profiles were assayed using a FACScan flow cytometry, and data were analyzed using MultiCycle software (both from BD Biosciences). Cell apoptosis was measured using the PE Annexin V apoptosis detection kit (BD Biosciences). After 48 h of culture, the cells were harvested and processed as described in the Annexin V-PE apoptosis detection kit and analyzed on a FACScan flow cytometry.

Western blotting was carried out on whole-cell extracts which were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100 and 1% sodium deoxycholate, 0.1% SDS) that contained protease inhibitors for 20 min at 4°C. The proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk in PBS-Tween-20 for 1 h at room temperature, the membranes were incubated with primary antibodies targeting β-actin, survivin, cyclin D1 and PARP (Abcam Inc., Cambridge, MA, USA). After washing three times with TBST, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibody (Beyotime Biotechnology) for 2 h. The proteins were visualized using enhanced chemiluminescence (ECL; Beyotime, Nantong, China).

Results are expressed as means ± standard error of the mean (SEM). Statistical analyses were performed using SPSS 17.0 software (IBM, Armonk, NY, USA). P-value <0.05 was considered to indicate a atatistically significant result.

To investigate REV3L gene expression in ESCC and adjacent tissues, the mRNA level of REV3L was analyzed by qRT-PCR using 68 ESCC and 48 adjacent tissues. As shown in Fig. 1a, the expression of REV3L in the ESCC tissues was significantly higher than that in the adjacent tissues (P<0.05). To further evaluate the correlation of REV3L mRNA expression and clinicopathological features, the characteristics of 68 ESCC patients included in the present study were analyzed (Table I). As shown by Mann-Whitney U test, REV3L mRNA expression was positively correlated with lymph node metastasis (Fig. 1B; P<0.05) and clinical stage (IIb and III) (Fig. 1C; P<0.05). In contrast, REV3L mRNA expression was not correlated with pT, gender, age and histological grade among the groups of patients. Collectively, these findings indicate that overexpression of REV3L in ESCC is associated with lymph node metastasis and tumor progression.

To study the potential role of REV3L in esophageal cancer, stable cell lines with REV3L knockdown were established using the ECA-109 and TE-1 cells. REV3L mRNA and protein expression levels were analyzed by RT-PCR and immunofluorescence to verify the successful knockdown of REV3L in these cells. The results revealed that REV3L expression at the mRNA and protein levels was significantly lower (P<0.05) in the cells stably transfected with the shREV3L construct as compared to the cells transfected with the control vector (shNC), indicating effective knockdown of REV3L expression (Fig. 2A and B).

To examine whether the modulation of REV3L expression affects the tumorigenic properties of the esophageal cancer in vitro, we measured the abilities of cell proliferation and invasion using CCK-8 assay and transwell analysis, respectively. As shown in Fig. 3, the cell proliferation (Fig. 3A) and invasion (Fig. 3B) were both decreased in the REV3L-knockdown cells (TE-1/shREV3L and ECA-109/shREV3L cells) compared to the control cells (TE-1/shNC and ECA-109/shNC cells) (P<0.05). These results suggest that REV3L plays an important role in esophageal cancer progression.

To further identify the mechanisms by which the silencing of REV3L inhibited esophageal cancer cell proliferation and invasion, we analyzed the expression levels of cyclin D1 and survivin proteins due to their established roles in cell proliferation and invasion. Western blot analysis revealed that REV3L knockdown significantly downregulated cyclin D1 and survivin protein expression in the esophageal cancer cells (Fig. 3C).

To study the possible role of REV3L in affecting the sensitivity of esophageal cancer cells to 5-FU, several concentrations of 5-FU were used to treat ECA-109 and TE-1 cells for 48 h. The cell viability was determined by the CCK-8 assay. As shown in Fig. 4, a dose-dependent inhibition in cell growth was observed in the 5-FU-treated TE-1 and ECA-109 cells, and the REV3L-knockdown cells were more sensitive to the cytotoxic effect of 5-FU. The results from the cell cycle analysis using PI staining showed that the number of cells in the G1 phase in the ECA-109/shREV3L cells was significantly higher than the number in the ECA-109/shNC cells after treatment with different doses of 5-FU for 24 h (Fig. 5). Together, these results indicate that REV3L plays a role in esophageal cancer resistance to 5-FU.

Next, we investigated whether the increased 5-FU cytotoxicity observed in the REV3L-knockdown cells was related to apoptosis. The extent of apoptosis was determined by measuring the percentage of Annexin V stained cells, which detect both early- and late-stage apoptosis. As shown in Fig. 6A, the apoptotic rates in the ECA-109/shREV3L cells were significantly higher than the rates in the ECA-109/shNC cells in response to different doses of 5-FU for 48 h. We then performed western blotting to analyze the cleavage of PARP protein, another established marker of apoptosis. As shown in Fig. 6B, ECA-109 REV3L-knockdown cells exhibited increased PARP cleavage compared with the control cells after treatment with 50 or 100 µM 5-FU for 48 h.