A tissue microarray containing 97 PDAC specimens, PDAC tumor tissue and paired adjacent tissue were collected from 97 patients with primary PDAC (age range, 21–77 years; sex, 60 males and 37 females) who underwent surgical resection from September 2011 to December 2013 at The Second Affiliated Hospital of Chongqing Medical University. All patients had been diagnosed with typical PDAC by the pathologist after surgery. TNM stage and clinical stage were evaluated according to the American Joint Committee on Cancer manual (17). The present study was approved by the Ethics Committee of The Second Affiliated Hospital of Chongqing Medical University and was conducted in accordance with the Declaration of Helsinki. An informed consent document was signed by all patients.

PDAC cell lines (CFPAC-1, HPAC, SW1990 and Capan-2), and pancreatic ductal epithelial cells (HPDE) were obtained from the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences). All cell lines were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), and were incubated at 37°C with 5% CO₂. A total of 3×10⁵ CFPAC-1 cells were transfected with 50 nM/ul siRNA (si) against HDAC9 (si-HDAC9) or negative control (si-NC). The sequences for the siRNAs were as follows: si-HDAC9 forward, 5′-AACGCCGGAGCUUUCACGTAT-3′ and reverse, 5′-GCGTTCAAUCAUGGUGGCACUTT-3′; and si-NC forward, 5′-AACUCCAAATGUTTCUCGATT-3′ and reverse, 5′-GAAUCACGCCCUAAGTTAATT-3′. The cells were transiently transfected using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc), and the transfection effect was maintained for >72 h.

Total protein was extracted from 1×10⁶ CFPAC-1, HPAC, SW1990, Capan-2 and HPDE cell lines, and 100 mg PDAC and adjacent tissues using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using the Bradford protein assay (Bio-Rad Laboratories, Inc.) with a Nanodrop spectrophotometer and 25 µg protein/lane was separated by SDS-PAGE on a 10% gel. After blocking with 5% non-fat powdered milk at room temperature for 1 h, the PVDF membranes were probed at 4°C overnight using the following primary antibodies: Anti-HDAC9 (1:1,000; cat. no. ab59718; Abcam), anti-Bax (1:5,000; cat. no. ab32503; Abcam), anti-Bcl-2 (1:500, cat. no. ab182858; Abcam), anti-KI67 (1:1,000; cat. no. ab15580; Abcam) and anti-GAPDH (1:5,000; cat. no. ab181602; Abcam). Subsequently, the PVDF membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. ab6721; Abcam) at room temperature for 1 h. Protein bands were visualized using enhanced chemiluminescence solution (EMD Millipore) and a ChemiDoc Imaging System (Bio-Rad Laboratories, Inc.). Protein expression was quantified using Quantity One version 4.6.6 software (Bio-Rad Laboratories, Inc.), with GAPDH as the loading control.

Total RNA from 1×10⁶ CFPAC-1, HPAC, SW1990, Capan-2 and HPDE cell lines, and 70 mg PDAC and paired adjacent tissues was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A PrimeScript RT kit (Takara Bio, Inc.) was used for RT of cDNA. The temperature protocol for RT was as follows: 32°C for 10 min, followed by 42°C for 30 min and 75°C for 10 min. qPCR was performed using SYBR^® Premix Ex Taq™ II (Takara Bio, Inc.) using a LightCycler system (Roche Molecular Systems, Inc.). The following primer sequences were used for the qPCR: HDAC9 forward, 5′-GAACTCTAAGCCAGATGGGG-3′ and reverse, 5′-GCCCACAGGAACTTCTGACT-3′; and GAPDH forward, 5′-TTCCAGCCTTCCTTCCTGGG-3′ and reverse, 5′-TTGCGCTCAGGAGGAGCAAT-3′. The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 2 min; 40 cycles of 95°C for 15 sec, 60°C for 34 sec and 72°C for 30 sec. The mRNA expression levels of HDAC9 were quantified using the 2^−ΔΔCq method (18) and expression levels were normalized to the internal reference gene GAPDH.

IHC of HDAC9 expression levels was performed using PDAC tissue microarrays. PDAC and paired adjacent tissues were fixed in 10% neutral formalin solution at room temperature for 24 h. Subsequently, paraffin-embedded tissue-array sections (4 µm) were dried at 80°C for 24 h, de-paraffinized in xylene I for 15 min and xylene II for 15 min, and then rehydrated in graded ethanol (100% ethanol for 5 min, 95% ethanol for 5 min, 80% ethanol for 5 min and 75% ethanol for 5 min). To block the endogenous peroxidase activity, the sections were incubated in 3% H₂O₂ for 30 min at room temperature. After washing with 0.01 M PBS three times, sections were incubated for 15 min at room temperature with 5% goat serum (OriGene Technologies, Inc.) to block non-specific binding, followed by incubation with a rabbit monoclonal anti-HDAC9 antibody (1:500; cat. no. ab109446; Abcam) at 4°C overnight. The sections were then incubated with an anti-rabbit secondary IgG antibody (1:5,000; cat. no. TA140003; OriGene Technologies, Inc.) at 37°C for 30 min. After washing with PBS, the signal was visualized using diaminobenzidine (Wuhan Boster Biological Technology, Ltd.), and counterstaining was performed with hematoxylin for 2 min at room temperature. The histopathological examination was performed using an Olympus DP70 light microscope (magnification, ×200; Olympus Corporation). Finally, HDAC9 immunostaining was scored and examined by two independent assessors, who were blinded to the clinicopathological data.

The staining intensity score and the proportion of HDAC9 positive cells were evaluated by the pathologist as follows: Staining intensity, i) negative=0; ii) weakly stained=1; iii) moderately stained=2; and iv) strongly stained=3. Staining extent: i) none=0; ii) 1–20%=1; iii) 21–40%=2; iv) 41–60%=3; v) 61–80%=4; and vi) 81–100%=5. The final immunoreactive score (IRS) of HDAC9 expression level was calculated by multiplying the staining intensity score with the staining extent score. IRS was dichotomised using X-tile software version 3.4.7 software (Yale School of Medicine), which is a useful bio-informatics tool for outcome-based cut-point optimization (19). IRS ≤7.5 was designated as low expression, while IRS >7.5 was designated as high expression.

A total of 2×10³ CFPAC-1 cells/well were transfected with si-HDAC9 or si-NC for 48 h and cultured in a 96-well plate for 24, 48 and 72 h. Cell proliferation analysis was performed using the CCK-8 assay (Dojindo Molecular Technologies, Inc.) according to the manufacturer's protocol.

A total of 3×10⁵ CFPAC-1 cells were transfected with si-HDAC9 or si-NC for 48 h and seeded into 6-well plates. When the cell density reached 70–80%, all cell lines were cultured in RPMI-1640 medium containing 0% FBS and were incubated at 37°C with 5% CO₂ for 24 h. The cell monolayer was scratched with a pipette tip (size, 10 ml) to generate three scratch wounds and then rinsed twice with PBS to remove non-adherent cells. Cells were visualized and counted using a light microscope (magnification, ×200). The distance between scratches was measured at 0, 24 and 48 h. The cell migration rate (%) was calculated using the following equation: [(Original gap distance-current gap distance)/original gap distance] ×100.

A total of 1×10³ CFPAC-1 cells were transfected for 48 h and cultured in a 96-well plate. Cells were incubated with 50 µM EdU, 100 µl 1X ApolloR reaction cocktail (cat. no. 100T; Guangzhou RiboBio Co., Ltd.) and 100 µl 1X Hoechst 33342 for 30 min at 37°C. Cell proliferation was analyzed by counting the mean number of cells in three fields for each sample using a fluorescence microscope (magnification, ×100).

A total of 6 BALB/c-nu mice (age, 5 weeks; sex, male; weight, 20–22 g) were purchased from The Shanghai Experimental Animal Center and housed in a sterile room at The Animal Center of Chongqing Medical University at 25°C and 40–70% humidity, with a 12-h light/dark cycle and free access to food and water. The study was approved by Ethics Committee of Chongqing Medical University (approval no. IACUC-20180117021). All animal experiments were performed in accordance with the institutional guidelines, and the method of euthanasia was cervical dislocation (when the heart stopped completely, the mouse was determined as dead). Body weight loss >20% was assumed to be a humane endpoint for euthanasia. Xenograft tumors were generated by subcutaneously injecting 3×10⁶ PDAC cells into the left hip flanks of the mice (n=3 per group; 2 groups; each mouse was inoculated with a single tumor site of PDAC cells). Tumor volume was calculated according to the following formula: Volume=(length × width²)/2. Then, 28 days after injection, the mice were sacrificed, and tumors were collected for analysis. The tumor experiments were ended when tumor diameters were <20 mm (the maximum tumor volume was 523 mm³).

Data are presented as the mean ± SD and each cell experiment was repeated three times. Statistical data were analyzed using SPSS version 23.0 software (IBM Corp.) and GraphPad Prism version 7.0 software (GraphPad Software, Inc.). A paired t-test was used to evaluate the significance of HDAC9 expression level between PDAC tissues and adjacent normal tissues. Association analyses of clinicopathological factors was performed with Pearson's χ² test. The Kaplan-Meier method was used to plot survival curves and calculate survival probabilities for overall survival. The statistical significance of survival curves was determined using the log-rank test. Univariate and multivariate analyses (Cox regression analysis) were applied to identify the clinicopathological features, and covariates with P<0.05 in univariate analysis were further analyzed by multivariate analysis. Statistical differences were analyzed by one-way ANOVA, followed by Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

To analyze the expression of HDAC9 protein in PDAC, RT-qPCR and western blot analyses were performed on PDAC and paired adjacent tissue. The expression of HDAC9 was significantly higher in PDAC tissues compared with paired adjacent tissues (Fig. 1A and B). Moreover, the expression level of HDAC9 was higher in PDAC cell lines compared with a pancreatic ductal epithelial cell line (Fig. 2).

The expression level of HDAC9 in PDAC was analyzed by IHC, and the results indicated that high HDAC9 expression was observed in 64.0% of PDAC samples (62/97). Furthermore, positive staining for HDAC9 was primarily localized in the nuclei of tumor and normal pancreatic cells (Fig. 3).

The present study analyzed the association between HDAC9 expression level and the clinicopathological characteristics of patients with PDAC. High HDAC9 expression level was identified to be positively associated with tumor size (P=0.026), T stage (P=0.014) and N stage (P=0.004; Table I). There was no significant association between HDAC9 expression level and age, sex, pathological grade, M stage, clinical stage or diabetes.

Kaplan-Meier survival analysis revealed that patients with PDAC exhibiting high HDAC9 expression levels have significantly shorter RFS compared with patients with low HDAC9 expression levels (P=0.017; Fig. 4A). To determine whether HDAC9 expression was an independent predictive factor for RFS in PDAC, Cox proportional hazards regression analysis was conducted. The univariate analysis indicated that T stage (HR=1.684; P=0.029), N stage (HR=1.448; P=0.013) and HDAC9 expression level (HR=1.647; P=0.018) were significantly associated with RFS in PDAC (Table II). The multivariate analysis suggested that T stage (HR=1.537; P=0.021), N stage (HR=1.597; P=0.019) and HDAC9 expression (HR=1.739; P=0.011) were independent prognostic factors for RFS in PDAC (Table II).

Kaplan-Meier survival analysis revealed that patients with PDAC exhibiting high HDAC9 expression levels had significantly shorter DSS compared with those with low HDAC9 expression levels (P=0.022; Fig. 4B). The univariate analysis suggested that T stage (HR=1.509; P=0.015), N stage (HR=1.493; P=0.025) and HDAC9 expression levels (HR=1.210; P=0.010) were significantly associated with DSS in PDAC (Table III). The multivariate analysis indicated that T stage (HR=1.805; P=0.011), N stage (HR=1.690; P=0.022) and HDAC9 expression levels (HR=1.394; P=0.008) were independent prognostic factors for DSS in PDAC (Table III).

After treatment with si-HDAC9, the expression level of HDAC9 was significantly decreased (Fig. 5A). A CCK-8 assay identified that decreased HDAC9 expression significantly inhibited cell proliferation in CFPAC-1 cells (Fig. 5B). Wound healing assay results indicated that CFPAC-1 cell mobility was significantly decreased following HDAC9 knockdown (Fig. 5C). The number of CFPAC-1 cells incorporating EdU in the HDAC9 knockdown group was significantly decreased compared with the control group (Fig. 5D).

The present study investigated the influence of HDAC9 in vivo. The results indicated that reducing the expression of HDAC9 may significantly inhibit the volume and weight of tumors formed by CFPAC-1 cells (Fig. 6A-C). In addition, key molecules involved in cell proliferation and apoptosis were analyzed. The present results suggested that HDAC9 silencing decreased Bcl-2 and Ki67 expression levels, and increased Bax expression (Fig. 6D).