A total of three PDAC datasets [GSE15471 (27), GSE16515 (28) and GSE32676 (29)] were obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The raw probe-level data (CEL files) and annotations for the probe arrays downloaded from GEO were used for analysis conducted by R software (version 3.5.0). The robust multi-array average algorithm in the Affy package of R was used for data pre-processing including background correction and quantile normalization. Then the probe ID was transformed to gene symbols. For multiple probe sets corresponding to the same gene, their average expression value was taken as the gene expression value.

A total of 79 pairs of PDAC tissues and adjacent non-cancerous tissues were collected from patients who underwent surgical resection at Ruijin Hospital Affiliated with Shanghai Jiao Tong University School of Medicine (Shanghai, China) between January 2011 and December 2017. All patients were diagnosed with PDAC via pathological diagnosis and had not received chemotherapy or radiotherapy before resection. The present study was approved by the Ethics Committee of Ruijin Hospital, and informed consent was obtained from all patients included in the study.

The human pancreatic cancer cell lines BxPC-3, MIA PaCa-2, PANC-1 and SW1990 were purchased from the Cell Bank of the Chinese Academy of Sciences. Capan-1, Capan-2 and the normal human pancreatic ductal cell line hTERT-HPNE were purchased from the American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Lonsera Science) and 1% penicillin/streptomycin at 37°C with 5% CO₂. LY294002 was purchased from Absin (cat. no. abs810001).

A TMA including 79 pairs of matched PDAC tissues and adjacent non-cancerous tissues was analyzed. The immunohistochemical procedure was described in our previous study (30). An antibody against DEPDC1B (1:100; cat. no. PA5-72875; Invitrogen; Thermo Fisher Scientific, Inc.) was used to detect the protein expression levels according to the standard procedure (30). The final IHC score was calculated by multiplying the percentage of positive cells (0, ≤5%; 1, 5–25%; 2, 25–50%; 3, 50–75%; 4, >75%) by the intensity scores (0, negatively stained; 1, weakly stained; 2, moderately stained; 3, strongly stained). Patients with a score ≥4 were regarded as having high expression of DEPDC1B. In contrast, patients with an IHC score <4 were described as having low expression.

Total RNA of hTERT-HPNE and pancreatic cancer cells was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 2 µg of total RNA was used to synthesize cDNA with the First Strand cDNA Synthesis SuperMix for RT-qPCR (Shanghai Yeasen Biotechnology Co., Ltd.), according to the manufacturer's protocol. Then, qPCR reactions were performed using SYBR Green Master Mix (Shanghai Yeasen Biotechnology Co., Ltd.) on a StepOnePlus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Relative mRNA expression was analyzed using the 2^−ΔΔCq method and normalized to GAPDH (31). RT was performed at 42°C for 15 min, followed by 2 min at 85°C. The qPCR program used included an initial denaturation at 90°C for 5 min, followed by 40 cycles of denaturation at 90°C for 10 sec and annealing/elongation at 60°C for 30 sec. The primers used were as follows: DEPDC1B: Forward, 5′-CCACCAGGCACTTCACAAGAGAAC-3′; and reverse, 5′-CGGTGGACAAGACGGCAAGC-3′; GAPDH: Forward, 5′-CAGGAGGCATTGCTGATGAT-3′; and reverse, 5′-GAAGGCTGGGGCTCATTT-3′.

Total protein was isolated from hTERT-HPNE and pancreatic cancer cells using RIPA containing 1% PMSF (both from Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Protein concentrations were determined using the BCA Protein Assay kit (Beyotime Institute of Biotechnology). Protein samples (30–60 µg per lane) were separated with 10% SDS-PAGE gels and transferred to PVDF membranes (EMD Millipore). Then, the membranes were blocked in TBS-Tween-20 containing 5% fat-free milk for 2 h at room temperature and incubated with the primary antibodies overnight at 4°C with gentle shaking. The primary antibodies included DEPDC1B (1:1,000; cat. no. PA5-72875; Invitrogen; Thermo Fisher Scientific, Inc.), E-cadherin (1:1,000; cat. no. 3195T), N-cadherin (1:1,000; cat. no. 131116T), Vimentin (1:1,000; cat. no. 3932S), Snail (1:1,000; cat. no. 3879S), Slug (1:1,000; cat. no. 9585T), Akt (1:1,000; cat. no. 4691S), and phosphorylated (p)-GSK-3β (1:1,000; cat. no. 5558S) (all from Cell Signaling Technology, Inc.), Twist (1:1,000; cat. no. ab49254; Abcam), p-Akt-S473 (1:1,000; cat. no. AP0140; ABclonal Biotech Co., Ltd.), GSK-3β (1:1,000; cat. no. A16868; ABclonal Biotech Co., Ltd.), and GAPDH (1:2,000, cat. no. GB12002, Servicebio; Stratech). After washing with TBST three times, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. no. 7074P2 and 7076S; Cell Signaling Technology, Inc.) at room temperature for 1 h. Signals were detected using enhanced chemiluminescence regent (GE Healthcare Bio-Sciences).

Validated small interfering (si)RNA targeting human DEPDC1B (5′-TGCTAGATTGGTAACGTTT-3′) and its corresponding negative control (NC; 5′-TTCTCCGAACGTGTCACGT-3′) were synthesized and inserted into the lentiviral vector GV248 by GeneChem, Inc. For DEPDC1B overexpression, the coding sequence (CDS) of DEPDC1B was amplified and cloned into pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO vector. The empty vector was used as the negative control for the overexpression assay. Recombinant lentiviruses overexpressing DEPDC1B and the negative control were provided by Hanbio Biotechnology Co., Ltd.

For the inhibition and overexpression of DEPDC1B, PDAC cells (Capan-1, SW1990, MIA PaCa-2 and PANC-1) were cultured to 30–50% confluence and transfected with lentivirus and polybrene overnight at the MOI (multiplicity of infection) of 10–30 according to the manufacturer's protocol. Stable transfected cells were selected in culture medium containing puromycin (2 µg/ml) for 10 days.

To measure cell viability, the transfected pancreatic cancer cells were seeded in 96-well plates (1×10³ cells/well). A total of 100 µl complete culture medium containing 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology) was added to each well at different time points of interest. After incubation in the dark at 37°C for 2 h, the absorption at 450 nm was detected. Each group had five duplicate wells and the experiments were repeated in triplicate.

Different cells were plated in 6-well plates (500 cells/well) in triplicate. Cells were maintained in culture for 14 days, and were then fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) at room temperature for 20 min followed by staining with crystal violet solution (Beyotime Institute of Biotechnology) at room temperature for 30 min. Images were captured with a camera and the colonies were counted manually to access cell proliferation ability.

Capan-1, SW1990, MIA PaCa-2 and PANC-1 cells were seeded into 6-well plates at a density of 5×10⁵/well and maintained in culture until 100% confluent. At that point, wounds were made using a standard 200 µl pipette tip. Cells were washed with PBS to remove debris and cultured in DMEM without FBS. Images were captured at 0 and 48 h after scratching with a light microscope (magnification, x200; Nikon Corporation). The cell migratory ability was evaluated by measuring the percentage of wound healing. The 48 h wound healing percentage=(0 h area of wound-48 h area of wound)/(0 h area of wound).

Transwell chambers (Corning, Inc.) precoated with or without Matrigel (BD Biosciences) for 30 min at 37°C were used for invasion and migration assays, respectively. A total of 600 µl of DMEM containing 10% FBS was added to the lower chambers, and 5×10⁴ cells suspended in 200 µl of serum-free DMEM were placed in the upper chambers. After 24 h in culture, migrated and invaded cells were fixed with 4% paraformaldehyde and stained with crystal violet staining solution at room temperature for 10 min. Cells in the lower chamber were removed using a cotton-tipped swab. Finally, the numbers of migrated and invaded cells in five randomly selected fields were counted under a light microscope (magnification, x200; Nikon Corporation).

Cells were seeded on glass coverslips and maintained in culture for 24 h. Then cells were fixed with 4% paraformaldehyde at room temperature for 15 min, permeabilized with 0.3% Triton X-100 at room temperature for 10 min and blocked with 0.3% BSA (Shanghai Yeasen Biotechnology Co., Ltd.) in PBS at room temperature for 30 min. Then, primary antibodies against E-cadherin (1:50; cat. no. 20874-1-AP), N-cadherin (1:100; cat. no. 66219-1-Ig) (both from Proteintech Group, Inc.) and Vimentin (1:100; cat. no. 3932S; Cell Signaling Technology, Inc.) were added and incubated overnight in a humidified chamber at 4°C. On the following day, Alexa Fluor 555-labeled Donkey Anti-Rabbit IgG (H+L) (1:200; cat. no. A0453; Beyotime Institute of Biotechnology) or IFKine™ Red Donkey Anti-Mouse IgG (1:200; cat. no. A24411; Abbkine Scientific Co., Ltd.) were applied and incubated for 1 h in the dark at room temperature. Finally, the nuclei of cells were counterstained with DAPI (Servicebio; Stratech) at room temperature for 5 min. All washes were performed with PBS. Images were captured with a fluorescence microscope (magnification, x400; Nikon Corporation).

A total of 10 female BALB/c nude mice (age, 4–5 weeks; body weight, 12–16 g) were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences and housed (5 mice per cage) under standard conditions (26°C, 40–60% relative humidity, 12 h light/dark cycles) with unlimited access to food and water. Animal health and behavior were monitored every day. For tumor metastasis assays, DEPDC1B knockdown and negative control Capan-1 cells (3×10⁶ cells) were intravenously injected into the tail vein of female 6-week-old nude mice (n=5 per group). After 10 weeks, the mice were euthanized in a gradually filled CO₂ chamber with a flow rate of ≤30% CO₂ of the chamber volume/min. The death was verified by the absence of respiratory movement and heartbeat. The lungs of the mice were dissected for H&E staining to assess the lung metastatic foci. All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai Jiaotong University School of Medicine (IACUC approval no. B-2019-004).

All data were analyzed using SPSS software (version 20.0; IBM Corp.) and are presented as the mean ± standard error (n≥3). Student's t-test was used to assess the statistical significance between two groups. One-way ANOVA followed by the Least Significant Difference post hoc test was used to compare multiple groups. The χ² test was used to analyze the association between DEPDC1B expression and clinicopathological parameters. Survival analysis was evaluated using the Kaplan-Meier method and log-rank test. P<0.05 was considered to indicate a statistically significant difference.

The three datasets (GSE15471, GSE16515 and GSE32676) were analyzed from the GEO database and the results showed that DEPDC1B mRNA was significantly upregulated in PDAC tissues compared with that in adjacent non-cancerous tissues (Fig. 1A). To further confirm this finding, immunohistochemical analysis were performed in a TMA containing 79 samples of PDAC and adjacent non-cancerous tissues (Fig. 1B). According to the IHC score, the expression levels of DEPDC1B were higher in PDAC tissues than those in paired non-cancerous tissues (Fig. 1C and D). No significant association between DEPDC1B expression levels and the clinicopathological characteristics of the samples was observed (Table I). However, Kaplan-Meier survival analysis revealed that patients with PDAC with higher expression levels of DEPDC1B could have a shorter survival time (Fig. 1E). Meanwhile, the data from The Cancer Genome Atlas also verified the association between high DEPDC1B expression and poor prognosis (Fig. 1F).

DEPDC1B expression was detected in six PDAC cell lines and the normal pancreatic cells hTERT-HPNE by RT-qPCR and western blotting. Higher expression of DEPDC1B was observed in PDAC cell lines compared with hTERT-HPNE (Fig. 2A and C). Capan-1 and SW1990 cells with high DEPDC1B expression, MIA PaCa-2 and PANC-1 cells with low DEPDC1B expression were selected for following experiments. Through a lentiviral delivery system, DEPDC1B was stably knocked down in Capan-1 and SW1990 cells and overexpressed in MIA PaCa-2 and PANC-1 cells. The transfection efficiency was confirmed by RT-qPCR and western blotting (Fig. 2B and D).

Considering the association between high DEPDC1B expression and poor prognosis, the role of DEPDC1B in cell proliferation, migration and invasion was further investigated. CCK-8 assay and colony formation assays revealed that cell proliferation was inhibited by DEPDC1B knockdown and promoted by DEPDC1B overexpression (Fig. 2E and F). Wound healing and Transwell assays were performed to investigate the role of DEPDC1B in cell migration and invasion. The wound healing assay showed that silencing DEPDC1B inhibited the migratory ability of Capan-1 and SW1990 cell lines, whereas DEPDC1B overexpression increased the wound healing rate of MIA PaCa-2 and PANC-1 cells (Fig. 3A). Transwell assays indicated that knockdown of DEPDC1B suppressed the migration and invasion of PDAC cells, while DEPDC1B overexpression promoted PDAC cell migration and invasion (Fig. 3B).

To further investigate the effect of DEPDC1B on tumor metastasis in vivo, a lung metastatic model was established in nude mice. The results demonstrated that the incidence of lung metastasis in the si-DEPDC1B group was lower than that in the control group (Fig. 4). The mean tumor diameter in the control group was 1.0 mm and the largest tumor measured 6.0 mm. No metastatic nodules were observed in the si-DEPDC1B group.

EMT is an important event in tumor metastasis, which allows cancer cells to acquire invasive properties and to develop metastatic growth characteristics (16). Therefore, the present study hypothesized that DEPDC1B may be involved in the EMT process. To investigate this effect, the expression of EMT markers and EMT-inducing transcription factors (EMT-TFs) was analyzed in the present study. Western blotting assays revealed that the expression of the epithelial marker E-cadherin was increased, whereas the mesenchymal markers Vimentin and N-cadherin, and the EMT-TF Snail were markedly decreased in DEPDC1B knockdown cells (Fig. 5). Conversely, downregulation of E-cadherin and upregulation of Vimentin, N-cadherin and Snail were detected in the cell lines overexpressing DEPDC1B (Fig. 5). There was no significant difference in the expression of Slug and Twist between the NC and treatment group. In addition, immunofluorescent staining was performed to analyze the protein expression of E-cadherin, Vimentin and N-cadherin. The results of the immunofluorescence assay further verified the alteration of EMT makers in DEPDC1B knockdown and overexpression cells (Fig. 6). These results indicated that DEPDC1B may be involved in the EMT process in PDAC cells.

As the main member of EMT-TFs, Snail can bind to the E-box area of E-cadherin and suppress its expression, thereby inducing EMT (32–34). Numerous studies have revealed that Akt can inhibit GSK3β activation and subsequently lead to the stabilization of endogenous Snail (24,25,35). The present study hypothesized that DEPDC1B may induce EMT via the Akt/GSK3β/Snail pathway. As presented in Fig. 7A, DEPDC1B overexpression significantly increased the levels of p-Akt at S473, p-GSK3β and Snail, whereas these protein levels were decreased in DEPDC1B knockdown cells. These findings suggested that DEPDC1B may enhance Snail expression via the Akt/GSK3β pathway. To further verify whether Snail upregulation was mediated by Akt, DEPDC1B overexpressing cells were treated with the Akt inhibitor LY294002. The results showed that LY294002 treatment reversed the upregulation of p-Akt, p-GSK3β and Snail induced by the overexpression of DEPDC1B in both PANC-1 and MIA PaCa-2 cells (Fig. 7B). In addition, the inhibition of Akt increased the protein expression levels of E-cadherin and decreased Vimentin and N-cadherin expression (Fig. 7B). These results suggested that DEPDC1B promoted EMT via the Akt/GSK3β/Snail signaling pathway.