This study included 92 pancreatic cancer samples, including tumors and adjacent non-tumor tissues. The pancreatic cancer tissues were obtained from surgical specimens from inpatients in our hospital from 2016 to 2020.

Cells in a 24-well plate were incubated with 200 μl of EDU medium (5 μM) for 2 h and then fixed with 50 μl phosphate buffer saline (PBS) containing 4% paraformaldehyde for 30 min at room temperature. Cells were then incubated with 50 μl of glycine (2 mg/ml) for 5 min, followed by a 10-min treatment of 200 μl of osmotic agent (PBS containing 0.5% Triton X-100), a 30-min treatment of 200 μl of IX Apollo staining solution at room temperature in the dark, and two to three rinses with 200 μl of osmotic agent (PBS containing 0.5% Triton X-100; 10 min each time). Cells were counterstained with 4′, 6 diamidino-2-phenylindole (DAPI) for 5 min for nuclear staining and then examined using a fluorescence microscope.

It is predicted that the human DNA sequences of the p21 promoter are bound with transcription element p53 cloned into a pGL3 vector. Cells were transfected with luciferase plasmids along with ZWINT or control lentivirus for x h. Cell lysates were obtained and luciferase activity was detected using the dual-luciferase reporter system according to the manufacturer’s instructions (Promega, Madison, WI, United States).

RNA was extracted from cells using Trizol based on the manufacturer’s instructions, and a UV spectrophotometer was used to determine the purity and concentration of the RNA. RNA was reverse transcribed into cDNA using the RT-PCR kit TAKARA047A (Takara Bio, Inc., Shiga, Japan) of the Super Script III First-Strand Synthesis System. The BioRad Real-Time PCR system was used for real-time PCR amplification. The primers for ZWINT, p53, p21, CDK4, CDK6, Cyclin D1, Cyclin E1, and GAPDH mRNAs are shown in Table 1.

Immunohistochemistry was performed on 5-μm-thick paraffin sections. Monoclonal antibody against ZWINT was used at a 1:50 dilution (HAP022264, Sigma). After dewaxing, immunostaining permeation solution was applied to the samples. The presence of brown particles in the nucleus and cytoplasm indicated positive staining.

The EZ-ChIPTM Chromatin Immunoprecipitation Kit (Millipore, Billerica, MA, United States) was used to perform ChIP assays following the manufacturer’s instructions. Rabbit anti-FLAG (Cell Signaling Technology, United States), anti-RNA polymerase II antibodies (Abcam, United Kingdom) and related rabbit-IgG (Cell Signaling Technology, United States) was applied as controls.

The pancreatic cancer cell lines (AsPC-1, BxPC-3, MIA PaCa-2, SW 1990, PANC-1 and PANC03.27) and the HPDE normal pancreatic cell line were purchased from the ATCC. MIA PaCa-2, SW 1990, and PANC-1 cells were cultured in conventional DMEM medium (GIBCO, United States) containing 10% fetal bovine serum (BI, United States). AsPC-1, PANC03.27 and HPDE cells were cultured in RPMI 1640 medium (GIBCO) containing 10% fetal bovine serum. Cells were cultured at 37°C, 5% CO₂ with saturated humidity.

The liposome method (Lipo 3000 transfection kit, Invitrogen) was used to transfect small interfering RNA (siRNA) into PANC-1 and MIA PaCa-2 cell lines (siRNA sequences were shown in Table 2). Cells were harvested at 48 h and analyzed by RT-PCR and western blotting to determine the effect of siRNA transfection. Table 2 shows the siRNA sequences of ZWINT, p53, p21, and that of the negative control (Ruibio, Guangzhou, China). The virus-mediated transfection and oncogene overexpression were applied to infect PANC-1 and MIA PaCa-2 cell lines. Cells were cultured in flasks at a density of 2×10⁴ cells/culture flask the day before infection. After overnight culture, the medium was replaced with DMEM (without serum) containing lentivirus (MOI = 25) at 37°C. After 8–12  h, the medium was replaced with complete medium.

Cells were lysed using RIPA buffer for 30 min at 4°C, followed by centrifugation at 12,000×g at 4 °C for 30 min. The BCA method was used to determine the protein concentration of supernatants. Denatured protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, United States). The membrane was blocked with 5% milk or bovine serum albumin (BSA) for 2 h and then incubated with primary antibodies at 4 °C overnight. After washes with Tris-buffered saline (TBST), membranes were incubated with secondary antibody (1:2000, abclone, Wuhan, China) for 2 h, followed by washes with TBST. The ECL reagent (Thermo Scientific ECL) was used to visualize bands. The experiment was performed three times.

Cell proliferation ability was assessed by CCK-8 (Dojindo Molecular Technologies, Inc., Kyushu, Japan) based on the manufacturer’s instructions. Cells were seeded into 96-well culture plates at 2×10³ cells per well the day before transfection or infection. The viability of pancreatic cancer cells was evaluated from five replicates in three independent experiments after indicated treatments for 6, 24, 48, and 72 h.

Cells were fixed with 70% ethanol overnight at 4 °C. A staining working solution of 500 μl PI/RNase (PI:RNaseA prepared at 9:1) (KeyGen Biotech, Nanjing, China) was added when centrifugation was used to remove the ethanol in the following day. Cells were stained for 3 min at 4°C in the dark. Stained cells were then examined using a flow cytometer (US BD company).

Anti-ZWINT antibody (1 μg) (Sigma: HAP022264) or anti-rabbit IgG antibody (Abcam: ab190492) was added to protein lysates (500 μg) and the samples were incubated at 4 °C overnight. Next, 40 μl protein A/G PLUS-Agarose beads (Santa Cruz: sc-2003) was added and samples were incubated at 4°C for 6 h. After adding 2 × loading buffer, the immunoprecipitated protein complex was boiled and denatured. Samples were then analyzed by western blot. 1 μg of the anti-ZWINT primary antibody was employed to perform the same approach, so as to precipitate p53 and MDM2, and western blotting with the anti-p53 and anti-MDM2 primary antibody was conducted to detect the precipitated protein.

Cells on glass coverslips were fixed with 4% paraformaldehyde for 20 min at room temperature. After 15 min incubation with immunostaining permeation solution (Triton X-100, P0096, Beyotime), PBS containing 5% BSA was used to block cells for 30 min. Cells were then incubated with primary antibody against ZWINT (1:50, diluted in blocking buffer) at 4 °C overnight and then with Alexa Fluor 488-goat anti-mouse IgG (1:100) secondary antibody for 2 h. Cells were then rinsed with PBS for 15 min, and DAPI was applied for 5 min for nuclei staining. In some experiments, cells were incubated with anti-p53 and anti-p21 primary antibody and the anti-ZWINT primary antibody (1:50, Sigma) simultaneously. Alexa Fluor 488-goat anti-mouse and Alexa Fluor 594-goat anti-mouse were used as secondary antibodies.

Twelve 4-week-old female BALB/cA-nu nude mice were obtained from Beijing Huafukang Biosciences (Beijing, China) and kept in pathogen-free conditions. PANC-1 cells (5 × 10⁶) expressing either ZWINT or control vector in 150 μl of PBS were subcutaneously injected into the left flank of mice (n = 5 per group). The rate of tumor formation, number of tumors, tumor diameter, tumor mass and mouse body weight were measured every 7 days. Tumor volume was calculated as V = 1/2 × L × W². Mice were killed after 37 days. Tumors were fixed in 4% paraformaldehyde and embedded in paraffin.

The x ² test and Fisher’s exact probability tests were used to analyze data, and the single-element analysis of variance was employed to measure the data. The t test was used to determine statistical variations between two groups, and more than two groups were compared using the one-way analysis of variance analysis. A two-tailed p value of <0.05 was regarded statistically significant.

To determine the role of ZWINT in pancreatic cancer tumorigenesis, a bioinformation analysis was performed using TCGA database. The results indicated that ZWINT was significantly upregulated in pancreatic cancer tissue (Figure 1A). We next performed qRT-PCR assay using 16 pairs of PC and adjacent non-tumor tissues and found that ZWINT mRNA was highly expressed in PC tissues (Figure 1B). ZWINT was further examined in a tissue microarray that included 92 paired PC samples. The average expression of ZWINT was increased in PC tissues compared with levels in nearby non-tumor tissues (Figure 1C). The positive ZWINT expression rate was 65.2% (60/92) in PC tissue samples. Further analyses of PC cell lines by qPCR and western blot assay demonstrated that ZWINT mRNA and protein level was upregulated in PC cell lines (Figures 1D,E). Based on the GEPIA (http://gepia.cancer-pku.cn/) and survival data from the cohort, Kaplan-Meier analysis, ROC analysis results indicated that ZWINT expression was negatively correlated with PC patient overall survival (Figures 1F,G). We further analyzed the relationship between ZWINT expression and clinicopathological features in PC tissue samples (Table 3). ZWINT expression was positively related to tumor size, differentiation and Vessel invasion in PC tissues. Together these results indicate that low ZWINT expression is related to progression and poor prognosis in human PC.

To clarify the role of ZWINT in regulating PC cell phenotype, we performed loss-of-function and gain-of-function assays in PC cells (Figures 2A,B). CCK-8 and colony formation assays showed that PC cell proliferation was reduced by ZWINT knockdown, while increased proliferation was observed in cells with overexpression of ZWINT (Figures 2C,D). EDU assay also showed that ZWINT overexpression significantly promoted PC cell proliferation (Figure 2E). We next evaluated the role of ZWINT in cell cycle regulation of PC cells using flow cytometry assays. We detected a significant increase in the G1/S phase population of ZWINT-depleted PC cells compared with the control group (Figure 2F). qPCR and western blot further demonstrated that ZWINT promoted cell cycle–associated gene and protein expression (Figures 2G,H). These findings indicate PC cell proliferation induces G1/S phase arrest was enhanced by ZWINT, making contribution to PC progression.

We next explored the elements that induced high ZWINT expression in PC. Promoter sequence analysis tools (UCSC and JASPAR) (Figure 3A) were used to inspect the genomic region (∼2 kb upstream) upstream of the ZWINT gene. The results showed two putative HIF1α binding sites within the ZWINT promoter area (Figure 3B). We next performed ChIP-qPCR assay and confirmed that HIF1α directly bound to the chromatin fragments of the two predicted ZWINT promoter areas (Figure 3C). Luciferase assays also demonstrated that wild-type (WT) ZWINT promoter activity was significantly upregulated by HIF1α, while the mutant construct with mutations in the putative HIF1α-binding sequences showed no changes in response to HIF1α expression (Figure 3D). We performed q-RT-PCR analysis for ZWINT and HIF1α mRNA in PC tissues and the pearson analysis results revealed a positive correlation (Figure 3E). After 24 h treatment of hypoxia or the chemical inducer CoCl₂, the expression of ZWINT in PANC-1 and MIA PaCa-2 cells was increased on basis of upregulated HIF1α expression in the mRNA and protein level (Figures 3F,G). Immunohistochemistry of HIF1α and ZWINT in PC tissues showed that HIF1α and ZWINT expressions were positively correlated (Figure 3H). Together these data indicate that ZWINT is transcriptionally upregulated by HIF1α in the hypoxic tumor micro-environment. Subsequently, functional experiments were performed to measure the proliferation of PC cells in the condition of hypoxia compared with normoxia. The results suggested that hypoxia-mediated ZWINT overexpression significantly promoted cell proliferation compared with normoxia (Supplementary Figure S1).

To elucidate the downstream signal pathway of ZWINT, we analyzed the potential targeted pathways using the TCGA database. The results indicated that ZWINT likely regulates the cell cycle, DNA replication, and the p53 signaling pathway (Figure 4A). As ZWINT showed cell cycle regulation activity and p53 is a key regulator of the cell cycle, we next examined whether ZWINT affected p53 expression and activity. We first performed qRT-PCR to analyze p53 and p21 mRNA expression. ZWINT had no effect on p53 mRNA expression level and p21 expression was significantly upregulated in the ZWINT knockdown group (Figures 4B,C). Western blot assay revealed that p53 and p21 were significantly downregulated in ZWINT-overexpressing pancreatic cancer cells (Figure 4D).

Our results showed that ZWINT regulated p53 protein level. As p53 protein levels are regulated by MDM2-mediated ubiquitination, we further examined whether ZWINT interacted with MDM2 and p53 to affect p53 ubiquitination. We first analyzed the interactions between ZWINT, p53, and MDM2. Immunoprecipitation and immunofluorescence analysis indicated that ZWINT binds with and co-localizes with p53 and MDM2 (Figures 5A,B). Subsequently, western blot analysis showed that MDM2 expression was increased by the overexpression of ZWINT, while MDM2 levels were decreased by ZWINT knockdown (Figure 5C). We next examined whether ZWINT affect p53 ubiquitination and stability. Protein stability assay indicated that ZWINT significantly inhibited p53 stability and ubiquitination assay demonstrated that p53 ubiquitination was significantly increased in the ZWINT-upregulated group (Figures 5D–F). These results suggest that ZWINT promoting the effect of MDM2 and influences the ubiquitination, stability and expression level of p53 by decreasing the expression level of MDM2.

To investigate whether p53/p21 is necessary for ZWINT regulation of PC cell proliferation and cell cycle, we transfected p53 and p21 siRNA into ZWINT knockdown PC cells. CCK-8, colony formation assay and EDU proliferation assays showed that proliferation in ZWINT knockdown cells was enhanced upon p53 or p21 silencing (Figures 6A–C). qRT-PCR and western blot assays showed that p53 or p21 inhibition in PC cells abolished the inhibition of the expression of p53 and p21 reduced by ZWINT knockdown (Figures 6D–G).

To determine whether tumor growth is affected by ZWINT in vivo, we established a xenograft mouse model by subcutaneously injecting pancreatic cancer cells overexpressing ZWINT or controls into mice (n = 5, each group). After 6 weeks, mice were killed and tumor samples were harvested (Figure 7A). Tumor weight (Figure 7B) and volume (Figure 7C) from cells with knockdown of ZWINT were reduced compared with controls. Immunohistochemistry showed that Ki-67 and PCNA were highly expressed in tumors from the ZWINT upregulated group and downregulated in the knockdown group (Figure 7D). Together these data indicate that ZWINT promoted pancreatic cancer growth in vivo.