Data analysis of public databases
To get the expression pattern of CKS2 in pancreatic cancer, the data sets were downloaded from a newly developed interactive web database Gene Expression Profiling Interactive Analysis (GEPIA: http://gepia.cancer-pku.cn/) based on the TCGA and GTEx projects for RNA expression analyses. Total 350 samples, including 171 normal cases and 179 tumor cases were obtained for CKS2 expression and the prognostic significance of CKS2 expression for the overall survival and disease-free survival in pancreatic cancer patients by Kaplan-Meier plotter. Disease-free survival was defined as the length of time following resection during which a patient survived without signs of recurrence or metastasis. Low and high expression of CKS2 in the pancreatic cancer was defined according to the median, and those above the median were defined as high expression and those under the median were defined as low expression. Meanwhile, the expression pattern of MKi67 was also obtained in the 179 tumor cases and Spearman’s correlation analysis was performed to assess the relationship between CKS2 and MKi67 in pancreatic cancer patient samples.
Clinical sample information
A total of 64 of paraffin-embedded tumor tissues and matched adjacent non-cancerous tissues (2 cm away from the tumor border) were obtained from our Hospital, with all pancreatic cancer patients signed written informed consent. Radiotherapy or chemotherapy was not received by any of these patients before the operation and follow-up continued up to November 2018. The basic clinical characteristics are summarized in Table S1. The overall survival (OS) is taken to indicate the time from surgery to death or final contact for any reason. The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Nanjing Medical University.
Immunohistochemical staining for CKS2 was performed using an ultrasensitive kit (MXB; Fuzhou, China) according to the manufacturer’s instructions. In brief, paraffin-embedded specimens were fixed with 10% buffered-formalin and made into 4 μm-thick sections. The sections were subsequently dewaxed in xylene and rehydrated in decreasing gradient ethanol. After rinsed in PBS, antigen retrieval was done with high-pressure steam treatment at 100 °C in 10 mM citrate buffer (pH, 6.0). Next, the endogenous peroxidase activity was blocked with peroxidase blocking solution for 10 min. Then, the sections were incubated with rabbit polyclonal anti-human CKS2 antibody (1:100; Santa Cruz Biotechnology) overnight at 4 °C, followed by probed for 2 h with horse-radish peroxidase (HRP)-conjugated secondary antibodies at 37 °C. Reaction product was developed with 3, 3'-diaminobenzidine (DAB) substrate kit. Two independent pathologists evaluated the immunohistochemical staining based on the proportion of staining area (0–100 scale) and the intensity of staining (0 = negative; 1 = weak; 2 = moderate; 3 = strong). Finally, these two scores were then multiplied together to yield an immunohistochemistry score between 0 and 300. All patients were divided into high- and low-expression groups with the mean immunohistochemistry score as a cutoff value.
Human pancreatic cancer cell lines, including BXPC-3, Mia PaCa-2, PANC-1, SW1990, COLO 357, CFPAC-1, and in immortalized non-neopastic pancreatic dual cell line hTERT-HPNE were provided by American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were cultured in RPMI1640 medium (Wako, Tokyo, Japan) with 10% heat-inactivated FBS (Gibco, Carlsbad, CA, USA) at 37 °C in a 5% CO2 incubator.
Lentiviruses carrying short hairpin RNA targeting CKS2 (Lv-sh-CKS2), negative control (NC), CKS2 (Lv-CKS2) or empty vector were produced by RiboBio (Guangzhou, China). Media containing lentivirus particles carrying Lv-sh-CKS2 or NC were used to transfect Mia PaCa-2 and SW1990 cells, while those carrying Lv-CKS2 or vector were used to transfect PANC-1 cells using Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA), followed by puromycin selection.
Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was isolated from cells using TRIzol reagent (Invitrogen) and 2 μg RNA from each specimen was used for complementary DNA (cDNA) synthesis with PrimeScript™ Reverse Transcription kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocols. RT-qPCR was conducted with SYBR® Green Master Mix (Takara Bio, Inc., Otsu, Japan) following the reaction parameters (denaturation at 94 °C for 30 sec, then 30 cycles of 56 °C for 30 sec and 72 °C for 90 sec, and final extension at 72 °C for 5 min). Primer sequences used in this study were listed as follows: CKS2: forward, 5′-CTTCGCGCTCTCGTTTCATT-3′ and reverse, 5′-CACCAAGTCTCCTCCACTCC-3′; GAPDH: forward, 5′-GTCGATGGCTAGTCGTAGCATCGAT-3′ and reverse, 5′-TGCTAGCTGGCATGCCCGATCGATC-3′. Relative expression level of CKS2 was calculated using the 2-ΔΔCt method with GAPDH as an endogenous control.
After 48 h transfection, cells were seeded at a density of 3,000 per well in a 96-well plate and incubated for consecutive five days. At each day, cells in each well were incubated with 10 µl CCK-8 reagent (Dojindo Laboratories) for 2 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad, Berkeley, CA, USA). Triplicates were prepared for each sample and means were calculated.
5-Ethynyl-2ʹ-Deoxyuridine (EdU) assay
After 48 h transfection, cells were placed at a density of 1 × 105 cells per well into 96-well plates and incubated with 100 μL of 50 μM EdU reagent (RiboBio, Guangzhou, China). Then, cells were washed with PBS and fixed with 4% polyformaldehyde for 30 min at room temperature, which were treated with Apollo staining solution and Hoechst 33342 for 30 min in dark. The stained cells were examined under a fluorescent microscope (magnification 20×) and the percentage of Edu-positive cells was calculated using Image J software. Triplicates were prepared for each sample and means were calculated.
After 48 h transfection, cells (500 cells per well) were seeded in six-well plates and cultured for two weeks until forming colonies. Visible colonies were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet for 30 min. Subsequently, colonies with more than 50 cells were photographed and counted in five randomly selected fields. Triplicates were prepared for each sample and means were calculated.
Cells were harvested after transfection for 48 h and reseeded on 6-cm dishes at a density of 3 × 105 cells per well. For cell cycle analysis, cells were fixed with 70% ethanol and washed with PBS twice. Then, cells were re-suspended and stained with 5 μL of 25 μg/mL propidium iodide (PI, Keygentec, Nanjing, China) for 30 min in the dark. The percentage of cell population at different phases was determined by flow cytometry (BD Biosciences, San Jose, CA, USA). For cell apoptosis analysis, cells were stained with 5 µL Annexin V-FITC and 5 µL PI solution in 1× binding buffer (Keygentec) for 15 min at room temperature in the dark. Apoptotic cells were quantified by flow cytometry (BD Biosciences). Triplicates were prepared for each sample and means were calculated.
Wound healing assay
Cell migratory ability was assessed by performing wound healing assay. Briefly, transfected cells were plated in 12-well plates and cultured until 80% confluence. Then, an artificial scratch wound was created on cell plate with a 200 μl pipette tip. The wound closure was recorded at 0, 24 and 48 h, respectively under a light microscope. Relative wound healing percentage was calculated at 24 h and 48 h relative to 0 h in different groups. Triplicates were prepared for each sample and means were calculated.
Cell migration and invasion was assessed using 24-well Transwell chamber (Corning, Corning, NY, USA) coated without or with diluted Matrigel (BD Biosciences). In brief, the upper chamber was supplemented with 600 μL of serum-free medium containing 3 × 104 cells, while the lower chamber was filled with medium containing 10% FBS as a chemoattractant. Following 24 h of incubation, cells that migrated or invaded to the lower chamber were fixed in 4% paraformaldehyde for 15 min and stained with crystal violet for 10 min. Stained cells were photographed (magnification 200×) and counted using a light microscope. Triplicates were prepared for each sample and means were calculated.
Xenograft tumor in vivo
Six-week-old male BALB/c nude mice (18-21 g) were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China) and maintained in a specific pathogen-free (SPF) animal facility. Four groups of PDAC cells were prepared prior to inoculation as follows: empty vector-transfected PANC-1 cells, Lv-CKS2-transfected PANC-1 cells, NC-transfected Mia PaCa-2 cells and Lv-sh-CKS2-transfected Mia PaCa-2 cells. Approximately 1 × 106 transfected above cells were subcutaneously injected into the left dorsal flank of the mice and these mice were accordingly divided into four groups with six mice in each group. After inoculation, the tumor length (L) and width (W) were measured at 7, 14, 21, 28 and 35 days with calipers and tumor volume were calculated using the equation (L × W2)/2. On day 35, the mice were sacrificed by cervical dislocation and the tumors were excised and weighed. Excised tumors were paraffin-embedded for histologically examination by immunohistochemistry using anti-human Ki-67 (1:100; Santa Cruz Biotechnology). All the animal experimental procedures were approved by the Ethics Committee of Nanjing Medical University.
Total protein from cells was extracted using RIPA lysis buffer (Pierce, Rockford, IL, USA) and protein concentration was measured by the BCA protein assay kit (Beyotime, Jiangsu, China). Equal amount protein samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto PVDF membranes. After blocked with 5% skimmed milk for 2 h, the membranes were incubated overnight at 4 °C with primary antibodies against CKS2, Bax, Bcl-2, Caspase-3, Cyclin B1, CDK1, Cyclin A, P53, P21, GADD45α, Cdc25c, Cyclin D, CDK2, CDK4, β-actin and GAPDH. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 2 h. Protein signals were visualized using enhanced chemiluminescence (Keygentec) with β-actin or GAPDH as the internal control.
All statistical analyses were performed using SPSS 20.0 (SPSS, Inc., Chicago, IL, USA). The Kaplan-Meier method was conducted for the effect of CKS2 on survival analyses and the log rank test was used to examine the differences in survival. The correlation of CKS2 expression levels with clinicopathological features was evaluated using chi-square test. Independent risk factors among pancreatic cancer patients were identified using the univariate and multivariate Cox proportional hazards model. All data are presented as the means ± SD. For continuous data from in vitro and in vivo experiments, significant differences were determined using Student’s t-test for two groups and one-way ANOVA for more than two groups. P values less than 0.05 were regarded as statistically significant.