The human BxPC-3 pancreatic adenocarcinoma cell line was purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), and maintained at 37°C in a 5% CO₂ atmosphere.

The present study was approved by the Institutional Research Ethics Committee of the People's Hospital of Xinjiang (Urumqi, China). Written informed consent was obtained from patients prior to enrollment in the present study. A total of 48 patients who were admitted in People's Hospital of Xinjiang between 2013 and 2015 were included in the present study. Patient characteristics are presented in Table I. Patients were diagnosed with pancreatic cancer based on histopathological examination. No patients received chemotherapy or radiotherapy prior to surgery. Cancer tissue and adjacent normal tissue (distance >10 cm from the primary tumor) samples were isolated from patients who underwent tumor resection, and were immediately frozen in liquid nitrogen and stored at −80°C. Tissue samples were fixed in 10% neutral-buffered formalin at room temperature for 24 h. Following fixation, tissue samples were dehydrated by immersion in increasing concentrations of alcohol. Alcohol was cleared with xylene and tissues were embedded in paraffin by heating to 60°C and allowed to harden at room temperature overnight.

For the production of the lentivirus, 1 µg control short hairpin (sh)RNA (cat no. sc-108060; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or SATB1-targeting shRNAs (cat no. sc-36460; Santa Cruz Biotechnology, Inc.) were co-transfected with 1 µg packaging plasmids (0.4 µg pMD2G and 0.6 µg psPAX2; Santa Cruz Biotechnology, Inc.) into human 293FT cells (80% confluent) in DMEM supplemented with 10% FBS, using Effectene transfection reagent (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. Lentiviral supernatants were collected 48 h post-transfection and filtered through a 0.45 µm filter to remove debris. BxPC-3 cells cultured in DMEM supplemented with 10% FBS (80% confluent) were transduced at room temperature with 500 µl of viral supernatants at a multiplicity of infection of 10, containing 4 µg/ml Polybrene transfection reagent (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 18 h, and resistant colonies were selected with 2 µg/ml puromycin (Sigma-Aldrich; Merck KGaA) for 7 days. Successful transduction was confirmed by western blotting. Control cells were transduced with control shRNA.

Total proteins were extracted from 200,000 BxPC-3 cells following shRNA transduction, using Laemmli SDS reducing buffer (50 mM Tris-HCl pH 6.8, 2% SDS and 10% glycerol) at 4°C, boiled and quantified using a bicinchoninic acid protein assay. Equal amounts (30 µg) of extracted protein samples were resolved by 8–10% PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 3% milk at room temperature for 1 h, incubated with primary antibodies against SATB1 (cat no. ab92307; 1:1,000; Abcam, Cambridge, UK) and GAPDH (cat no. 2118; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat no. sc-2030, Santa Cruz Biotechnology, Inc.) at a dilution of 1:5,000 at room temperature for 1 h. Protein bands were visualized by enhanced chemiluminescence using the SuperSignal West Pico or Femto Chemiluminescent Substrate kits (Thermo Fisher Scientific, Inc.). Blots were semi-quantified using ImageJ software version 1.41 (National Institutes of Health, Bethesda, MD, USA) (17).

BxPC-3 cells stably expressing control shRNA or SATB1 shRNA were seeded in DMEM supplemented with 10% FBS at a density of 10,000 cells/well in 6-well plates in triplicate on day 0. Cells were trypsinized and counted using a TC20 Automated Cell Counter (Bio-Rad Laboratories, Inc., Hercules, CA, USA) on days 0, 1, 2 and 3. Each experiment was performed twice using cells from different suspensions.

BxPC-3 cells stably expressing control shRNA or SATB1 shRNA were seeded in 6-well plates at a density of 1,000 cells/well and cultured in DMEM supplemented with 10% FBS at 37°C. Cells were cultured for 1 week and then washed three times with PBS, fixed in 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet for 30 min at room temperature. Subsequently, the colonies (diameter, >1 mm) were carefully washed with PBS until the background was clear and visualized under an optical microscope. Colony formation efficiency was calculated as the number of colonies divided by 1,000 and normalized to control shRNA infected cells using ImageJ software verson 1.41.

BxPC-3 cells (1,000 cells) stably expressing control shRNA or SATB1 shRNA were suspended in 0.375% Noble agar (Difco; BD Biosciences, Franklin Lakes, NJ, USA) in DMEM supplemented with 10% FBS and overlaid on 0.75% Noble agar in 24-well plates. Colonies were allowed to grow for 7–10 days in the growth medium. Colony formation efficiency was calculated according to the following formula: Colony formation efficiency=(mean number of colonies/well)/(number of seeded cells/well). Colonies with a diameter >0.1 mm were measured and counted, and the mean was used. Data was expressed as fold-change compared to cells expressing the control vector. GraphPad Prism software version 5 (GraphPad Software, Inc., La Jolla, CA, USA) was used for analysis.

A total of 10⁵ BxPC-3 cells stably expressing control shRNA or SATB1 shRNA were serum-starved overnight, suspended in DMEM and plated into the upper chambers of 8.0-µm pore Transwell inserts which were coated with 400 µg/ml Matrigel (BD Biosciences). A total of 500 µl medium supplemented with 10% FBS was added to the lower chambers as a chemoattractant. Cells were incubated at 37°C for 24 h. Non-migrated cells on the top of the membrane were removed using cotton swabs, and cells that had migrated to the lower membrane were stained with 6% glutaraldehyde/0.5% crystal violet solution at room temperature for 30 min. Experiments were performed in triplicate. Invaded cells were visualized under an optical microscope, and counted using ImageJ software (17), by averaging the number of stained cells/field of view in 5 random fields/chamber.

Paraffin-embedded tumor and adjacent normal 5-µm thick tissue sections were subjected to antigen retrieval by heating in a microwave at 100°C for 10 min in 0.1 M citric acid buffer (pH 6.0), deparaffinized in xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 1 h at room temperature, following permeabilization with ice-cold 100% methanol for 10 min at −20°C and rinsed in PBS for 5 min. Sections were then incubated with an anti-SATB1 antibody (1:200; cat no. ab92307; Abcam) at 4°C overnight. Following incubation with HRP-conjugated secondary antibodies (cat no. BA-1000; 1:200; Vector Laboratories, Inc., Burlingame, CA, USA) at room temperature for 1 h, the slides were developed in 0.05% 3,3′-diaminobenzidine (Vector Laboratories, Inc.) containing 0.01% hydrogen peroxide at room temperature for 1 min. As a negative control, sections were incubated with normal goat serum (Invitrogen; Thermo Fisher Scientific, Inc.), instead of primary antibodies, at 4°C overnight. The density of the staining was ranked as follows: 0, no staining; 1, mild staining; 2, moderate staining; and 3, intense staining. The extent of staining was scored as follows: 0, no positive cells; 1, positive cells cover <10% of total area; 2, positive cells cover 10–50% of total area; and 3, positive cells cover >50% of total area. The final staining score was obtained by multiplying the intensity score with the extent score. The samples were classified into 2 groups according to the final score: Low (0–4) and high (5–9).

Animal experiments were approved by the Institutional Animal Care and Use Committee of the National Cancer Center (Urumqi, China). BXPC-3 cells expressing control shRNA or SATB1 shRNA (3×10⁶ cells/injection) were subcutaneously injected into both flanks of 10 female nude mice (age, 6 weeks; weight, ~25 g). The mice were purchased from the Chinese University of Hong Kong (Hong Kong, China) and maintained in individually ventilated cages under a 12-h light/dark cycle at 20–22°C and 40–60% relative humidity with free access to food and water. Between days 8 to 26 post-implantation, tumor volumes were measured using a caliper according to the following formula: Tumor volume (mm³)=tumor length × (tumor width)²/2]. Data were expressed as the mean tumor volume ± standard deviation. Mice were sacrificed 26 days post-implantation by CO₂ inhalation.

Total RNA was extracted from cancer tissue and paired normal mucosal tissue samples isolated from 48 patients with pancreatic cancer using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total RNA (1 µg) was reverse transcribed into cDNA using RevertAid™ First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). The reaction volume was 20 µl and contained 1 µg RNA and 1 µl dT primer, 4 µl reaction buffer, 2 µl dNTP, 0.5 µl inhibitor and 0.5 µl reverse transcriptase. The temperature protocol was as follows: At 65°C for 10 min, at 25°C for 10 min, at 55°C for 30 min, and then at 85°C for 5 min. PCR was performed on cDNA using a ViiA™ 7 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with SYBR^® Premix DimerEraser™ (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. Each experiment was performed in triplicate. Primer sequences were as follows: SABT1, forward 5′-AAAAGAAATCGGACCACCAAGC-3′, reverse 5′-TGTGGTACGGAGCTGATCG-3′; and GAPDH, forward 5′-GGCCAAGGTCATCCATGACAA-3′ and reverse 5′-TCTTCTGACACCTACCGGGGA-3′. Thermocycling conditions were as follows: Initial denaturation at 95°C for 2 min, followed by 40 cycles at 95°C for 10 sec, and at 60°C for 30 sec, with an extension at 72°C for 30 sec. The specificity of the amplification products was confirmed by the exhibition of a singlet in the melting curve and gene expression was quantified according to the comparative Cq method (18).

Data are expressed as the mean ± standard deviation of 3 independent experiments. Data were analyzed using one-way analysis of variance (ANOVA) or mixed-factorial ANOVA, where appropriate. Multiple comparisons were then further investigated using Tukey post hoc test. P<0.05 was considered to indicate a statistically significant difference. Survival curves were constructed using the Kaplan-Meier method and compared using log-rank test. Statistical analyses were performed using SPSS software version 16 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA).

To examine the functions of SATB1 on pancreatic cancer cell growth, stable SATB1-knockdown BxPC-3 cells were established using RNA interference. As presented in Fig. 1, following transduction with 2 independent shRNAs targeting SATB1, the protein expression levels of SATB1 in BxPC-3 cells were significantly downregulated. Knockdown of SATB1 expression significantly inhibited the proliferation of BxPC-3 cells compared with control cells, as demonstrated by a cellular proliferation assay (Fig. 2A). In addition, BxPC-3 cells transduced with SATB1-specific shRNAs exhibited significantly suppressed colony formation capabilities (Fig. 2B), thus suggesting that SATB1 may enhance the proliferation and clonogenicity of pancreatic cancer cells.

Soft agar growth and Matrigel invasion assays were performed to evaluate the effects of SATB1 knockdown on the tumorigenic properties of pancreatic cancer cells. As presented in Fig. 3, following the downregulation of SATB1 expression, the anchorage-independent growth of BxPC-3 cells was significantly inhibited. In addition, the invasive capabilities of cancer cells were significantly reduced following SATB1 shRNA transduction, as demonstrated by the Matrigel invasion assay.

To investigate the effects of SATB1 on pancreatic cancer cell growth in vivo, a mouse xenograft model was established following the subcutaneous injection of BxPC-3 cells expressing control shRNA or SATB1 shRNA-1 into nude mice. Tumor growth was monitored for 26 days post-xenotransplantation. As demonstrated in Fig. 4, SATB1 downregulation significantly reduced the weight and growth rate of tumor xenografts in vivo. These findings suggested that SATB1 may serve a role in promoting tumor growth in vivo.

Pancreatic cancer tissue and matched non-cancerous adjacent tissue samples were isolated from 48 patients with pancreatic cancer. Semi-quantitative RT-PCR demonstrated that the mRNA expression levels of SATB1 were significantly upregulated in pancreatic cancer tissues compared with in matched control samples (Fig. 5A). In addition, IHC results revealed that pancreatic cancer tissues exhibited stronger SATB1 staining compared with non-cancerous tissue samples (Fig. 5B). The patient cohort was divided into low and high SATB1 expression groups according to the IHC scoring, and a Kaplan-Meier survival analysis was performed. As presented in Fig. 5C, patients with high SATB1 expression had significantly shorter overall survival times compared with patients with low SATB1 expression scores.