Pancreatic cancer tissue collection and cell culture

Human survival pancreatic cancer tissues (n = 97) and paired pancreatic tissues (n = 69) were collected during surgical resection at Beijing Chaoyang hospital. The experimental procedures of this study were approved by the ethics committee of Beijing Chaoyang hospital (No. 2019-S-243) and all patients signed an informed consent form. Pancreatic cancer cell lines (PANC-1, SW1990 and BXPC-3) and normal pancreatic cell line HPDE6-C7 were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum (Invitrogen Gibco) and maintained in a 37 °C, 5% CO2 incubator.

Immunohistochemistry (IHC) staining

Formalin-immobilized tissues were dewaxed in xylene, rehydrated in ethanol solution, incubated with 3% hydrogen peroxide to block endogenous peroxidase and nonspecific binding sites. The tissues were incubated with the primary anti-ZNF655 (1:200, Invitrogen, PA5-56183), anti-CDK1 (1:50, Sigma, HPA003387) antibody at 4 °C overnight and then with the secondary antibody HRP goat anti-rabbit IgG (1:200, Beyotime, A0208) at room temperature for 1 h. After incubation with peroxidase conjugated streptavidin and diaminobenzidine, hematoxylin was stained. The staining intensity was scored according to the criteria described in the literature [23]. The result greater than or equal to median values of IHC was defined as ZNF655 high expression, otherwise low expression.

Lentivirus transfection and cell infection

Three shRNAs against ZNF655 or CDK1 interference sequences were synthesized (shZNF655-1, 5'-GCCCAGGAAGCAGCAGGGTCA-3'; shZNF655-2, 5'-CACCGACATGGAACAGGGACT-3'; shZNF655-3, 5'-TCCAGTTTCAGTCTTTGGAGA-3'; shCDK1-1, 5'-TTCCATGGATCTGAAGAAATA-3'; shCDK1-2, 5'-AGACTAGAAAGTGAAGAGGAA-3'; shCDK1-3, 5'-ATGGAGTTGTGTATAAGGGTA-3') and the lowest expression interference sequence of ZNF655 or CDK1 was selected, respectively. The fragment was inserted into lentiviral vector BR-V108 with green fluorescent protein (GFP) (BIOSCIRES). The 293 T cells were co-transfected with 10 μg recombinant BR-V108 vector and virus packaging plasmid (7.5 μg pMD2.G and 5 μg pSPAX2) (BIOSCIRES) for 72 h. Meanwhile, the amplified sequence of ZNF655 (ZNF655) was synthesized and inserted into lentiviral vector LV-003 (BIOSCIRES) to construct overexpression of ZNF655. After that, 2 × 105 PANC-1 and SW1990 cells were cultured for 24 h and infected with lentivirus shZNF655, shCDK1 and ZNF655 (1 × 108 TU/mL) at a MOI (multiplicity of infection) of 10. Finally, the expression of GFP was observed under fluorescence microscope (OLYMPUS) 72 h after lentivirus infection.

RNA extraction and qPCR

PANC-1 and SW1990 cells RNA was purified with Trizol [24] and reverse transcribed into cDNA using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantification of mRNA expression levels of ZNF655 and CDK1 was accomplished by SYBR Green master mix (Thermo Fisher Scientific) by ABI Prism 7500 sequence detection system (Applied Biosystems) with normalization to the expression of GAPDH. The primer sequences were listed in Table S1.

Western blotting (WB) and Co-immunoprecipitation (Co-IP) assay

PANC-1 and SW1990 cells protein was purified with RIPA (Beyotime) and the concentration was detected using BCA Protein Assay Kit (Beyotime). The 20 μg/well total protein was subjected to 10% SDS–PAGE, transferred to PVDF membrane (Millipore), hybridized with corresponding primary antibody (Table S2) overnight at 4 °C, incubated with secondary antibody at room temperature for 2 h. After that, protein signal was visualized through chemiluminescence ECL kit (Thermo Fisher Scientific) and GAPDH as load control. ZNF655 and CDK1 protein–protein interaction was analyzed by Co-IP assay and the experimental procedures were performed as previously described [25].

MTT and cell counting assay

PANC-1 and SW1990 cells were cultured in 96-well plates at a density of 2000 cells/well. Cell viability was detected by MTT assay for 5 days [26] and cell proliferation ability was analyzed by drawing growth curve. The cells with GFP were identified with Celigo, photographed, counted, and the cell growth curve was plotted for 5 days.

Detection of cell cycle and apoptosis

PANC-1 and SW1990 cells were inoculated into 6-well plates (2 mL/well) for 5 days.

For apoptosis analysis, the cell precipitates were successively washed by precooled D-hanks (pH = 7.2~7.4) and 1×binding buffer (eBioscience), resuspended by 200 μL 1× binding buffer, stained with 10 μL Annexin V-APC (eBioscience) at room temperature in the dark for 15 min and detected by flow cytometry (Millipore). For cell cycle analysis, the cells were centrifuged for 5 min, the cell precipitates were eluted with precooled PBS (pH = 7.2~7.4), fixed with 70% ethanol for at least 1 h, stained with PI (Sigma) and monitored by flow cytometry.

Human apoptosis antibody array assay

PANC-1 cells protein was purified with RIPA (Beyotime) and the concentration was detected using BCA Protein Assay Kit (Beyotime). The concentrations of 43 kinds of human apoptotic markers in cell lysate were simultaneously detected in strict accordance with the instructions of the kit (Human Apoptosis Antibody Array–Membrane, Abcam). After that, the signal of apoptotic markers was visualized through chemiluminescence ECL kit (Thermo Fisher Scientific) and quantified by Image J software (National Institutes of Health).

Wound-healing assay

PANC-1 and SW1990 cells were cultured in 6-well plates (100 μL/well) at a density of 4000 cells/well. The specific experimental procedures followed the description in the literature [27]. In order to avoid the influence of cell proliferation on migration, the cells were placed in a serum-free medium and treated with mitomycin for 1 h before performing wound-healing experiments. A line wound was drawn by a pipette tip across the cell layer. The cells were washed with PBS, fixed with 3.7% paraformaldehyde (Corning) for 15 min, stained with 1% crystal violet (Corning) for 10 min. The cells were viewed under a microscope for image acquisition and Image J software (National Institutes of Health) was used to quantify the distance (μm) between the scratches at 0 h, 8 h and 72 h.

Transwell assay

PANC-1 and SW1990 cells at a density of 80,000 cells/well were cultured into Transwell chambers (24-well, 8-mm pore, Corning) for 24 h at 37 °C, of which 100 μL cell suspension in the inner compartment and 500 μL DMEM medium containing 30% FBS in the outer compartment. The non-invading cells on the upper chamber were removed, while the cells adhering to the Polycarbonate membrane were fixed with 4% precooled paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min at room temperature. Finally, the cells were photographed from five randomly selected fields under a 200× microscope.

Gene microarray

After PANC-1 cells infected with lentivirus shZNF655 and shCtrl, RNA was purified and sequenced through Affymetrix human Gene Microarray Prime View (Affymetrix Scanner 3000 scan) to recognize differentially expressed genes (DEGs). DEGs was selected with criterion of |Fold Change | ≥ 1.3 and false discovery rate (FDR) ≤ 0.05 and presented as a volcano plot and hierarchical clustering. Significant enrichment of DEGs in classical pathways, disease and function, and interaction networks was explored based on Ingenuity Pathway Analysis (IPA).

Dual-luciferase reporter assay

The CDK1 promoter region was amplified and the fragment was cloned into the luciferase reporter vector GL002 (Promega Madison, USA), named as GL002- CDK1. Mutant construct GL002-CDK1-Mut was generated by site-directed mutagenesis. Luciferase assay was performed as described previously [28]. Each experimental analysis was repeated three times.

Chromatin immunoprecipitation (Ch-IP) assay

According to the manufacturer’s instructions, Ch-IP assay was performed using the Simple ChIP® Enzymatic Chromatin IP Kit (Cat No, 9002 S, CST, USA). In brief, the cells were transfected with ZNF655-overexpressing vector or its empty vector and incubated for 48 h at 37 °C with 5% CO2. Subsequently, the cells were crosslinked with 37% formaldehyde followed by lysed in SDS buffer and sheared sonication to fragment the DNA. Afterwards, the sonicated chromatin was precipitated by incubating it with according antibody overnight at 4 °C. The Protein–DNA complexes were then purified and the purified DNA was dissolved in nuclease-free water followed by qPCR analysis using the primers of CDK1 promoter and SYBR Green I Master (Roche, USA).

In vivo xenograft model of mice

The experimental procedures performed on mice were approved by the Ethics Committee of Beijing Chaoyang Hospital and the Animal Protection Association. The total of 20 BALB/c nude SPF mice (18–22 g, 4–6 weeks) were obtained from Viton Lihua Laboratory Animal Technology Co., Ltd. The 500 μL SW1990 cells infected with lentivirus shZNF655 (n = 5) and shCtrl (n = 5) were subcutaneously injected into the right armpit of mice (1 × 107 cells/mouse), respectively. Tumor volumes of mice was monitored 7 days after injection and then collected once or twice a week. Notably, the tumor volume was calculated using the formula π/6 × L × W × W (L: longest dimension, W: dimension perpendicular to length). After 26 days, mice were sacrificed by intraperitoneal injection of 0.7% pentobarbital sodium at a dose of 10 μL/g, and the tumors were removed for taking photos and weighting. Finally, the signal intensity of ZNF655 and CDK1 in mice tumor tissues was determined by IHC staining.

Statistical analysis

All data were presented as means ± SD and P < 0.05 were considered statistically significant. Comparisons between different groups were conducted by Student’s t-test or ANOVA as appropriate. The clinical relevance between ZNF655 and clinicopathological characteristics of pancreatic cancer patients was analyzed by univariate and multivariate analyses and Kaplan–Meier method.