Data from the GEPIA database (http://gepia.cancer-pku.cn/) were used to determine the association between ZFP403 and PCa. A gene symbol (ZFP403) was entered into the ‘Enter gene name’ field. The ‘GoPIA!’ button was clicked to generate the expression profile of the input gene across all tumor and normal tissues, in the form of dot plots or body maps. The ‘Boxplot’ tab in the ‘Expression DIY’ was clicked, and the ‘PRAD’ option was selected under ‘Cancer name’; the results were then presented in box plots.

PCa and adjacent tissues were collected from 19 male patients (aged 55 to 75 years old) who underwent treatment at the Department of Urology, Cancer Hospital of the University of Chinese Academy of Sciences (Hangzhou, China) between November 2013 and July 2015. The study was approved by the Hospital's Committee for the Protection of Human Subjects. All patients signed written informed consent forms in accordance with the Declaration of Helsinki.

IHC was used to determine the level of ZFP403 expression in clinical PCa tissues. The experiment was performed as previously described (14). The data were obtained by semi-quantitative analysis and finally presented as cut-off values. The cut-off values were determined by the multiplication of the staining intensity score and positive area score. Staining intensity scores were defined as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. Positive area scores were defined as follows: 0, <25%; 1, 26–50%; 2, 51–75%; and 3, >75%.

The LNCaP, PC3, DU145, 22RV1 and RWPE-1 cell lines were purchased from the Chinese Academy of Sciences. Between December 5th and 12th, 2019, these cell lines were authenticated by the analysis of 21 autosomal short tandem repeat loci (Biowing Applied Biotechnology Co. Ltd.). RWPE-1 cells were cultured in K-SFM medium (Gibco; Thermo Fisher Scientific, Inc.), and the other cell lines were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.). DMEM contained 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), and the cells were maintained at 37°C in a humidified atmosphere (5% CO₂).

Lentiviruses were purchased from Shanghai GeneChem. Short hairpin (sh)RNA sequences specifically targeting ZFP403 (shRNA1, 5′-GAGCAUACAAUAUCCUUAU-3′; shRNA2, 5′-GGGUAUUAGCAGAUUGGAA-3′) were cloned into the hU6-MCS-CMV-Puromycin vector. An empty vector was used as the negative control. The cells were seeded (1×10⁵) into a 24-well plate (Wuxi NEST Biotechnology Co., Ltd.) and cultured at 37°C overnight. Lentivirus (1×10⁸ TU/ml, 10 µl) was mixed with Opti-MEM (500 µl; Gibco; Thermo Fisher Scientific, Inc.) and polybrene (5 µg/ml; Shanghai GeneChem), and then used to infect the cells. The medium was replaced after 12 h, and complete medium containing 1 µg/ml puromycin (Sigma-Aldrich; Merck KGaA) was used to establish cells in which ZFP403 had been stably silenced. After a week, the transfection efficiency was assessed by reverse transcription-quantitative PCR (RT-qPCR) and western blot.

The mRNA levels of ZFP403 in PCa cells were analyzed by RT-qPCR. According to the manufacturer's instructions, total RNA was extracted using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) and then reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit (Takara Bio, Inc.). The concentration of nucleic acid was quantified using a cell Imaging Multi-mode Reader (BioTek Instruments, Inc.). Subsequently, qPCR was performed with SYBR-Green (Bio-Rad Laboratories, Inc.) using the CFX96 Real-Time system (Bio-Rad Laboratories, Inc.). The thermocycling parameters were as follows: 95°C for 2 min; 40 cycles at 95°C for 20 sec, 58°C for 20 sec, and 72°C for 15 sec; from 65°C to 95°C, to rise 0.5°C every 5 sec. The relative expression level of ZFP403 was quantified using the 2^−∆∆Cq method (17). The specific primer sequences were as previously described: ZFP403 forward, 5′-ACAGGGTATTAGCAGATTGGAAC-3′ and reverse, 5′-TCATTGGTAACAATTACTTCTACAC-3′; and GAPDH forward, 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse, 5′-AGGGGCCATCCACAGTCTTC-3′ (14).

Total protein was extracted from PCa cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) and the concentration of protein was determined using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). The samples (50 µg per lane) were separated by 8 or 10% SDS-PAGE and then transferred onto PVDF membranes (EMD Millipore). After blocking with TBST containing 5% skim milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with the following primary antibodies as appropriate: GGNBP2 (1:250; cat. no. ab203104, Sigma-Aldrich; Merck KGaA); cdc2 (cat. no. 9116), cyclin B1 (cat. no. 12231), β-catenin (cat. no. 9562), MMP-2 (cat. no. 4022) and β-actin (cat. no. 4970) (all 1:1,000; all from Cell Signaling Technology, Inc.); cdc25kC (1:500; cat. no. sc-13138; Santa Cruz Biotechnology, Inc.); E-cadherin (cat. no. 1702–1) and vimentin (cat. no. 2862-1) (1:1,000; both from Epitomics, Inc.); and heparanase (1;500; cat. no. ab42817; Abcam). Goat anti-rabbit IgG (H + L)-HRP and goat anti-mouse IgG (H + L)-HRP (1:3,000; Bio-Rad Laboratories, Inc.) were used as secondary antibodies and incubated with the membrane at room temperature for 2 h. Protein bands were visualized using Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Inc.) (18).

In vitro colony formation ability and tumorigenicity were investigated by plate and soft-agar colony formation assays. For plate colony formation assays, 500 cells per well were seeded into 24-well plates and cultured at 37°C. After 14 days, the cells were fixed with 4% paraformaldehyde for 15 min, and then stained with freshly prepared crystal violet for 20 min (both at room temperature). For the soft-agar colony formation assay, cells (1×10³ per well) were plated in 0.35% low melting point agarose above a solidified 0.7%-agarose layer in a 24-well plate. The cells were cultured at 37°C and the medium was changed twice a week. After 28 days, 1 mg/ml iodonitrotetrazolium chloride (Sigma-Aldrich; Merck KGaA) was added to each well and the plate was incubated for a further 24 h. The colonies were counted under a Leica DM500 microscope (magnification, ×40; Leica Microsystems, Inc.) (14).

The cells were stained using the PI/RNase staining kit (BD Pharmingen; BD Biosciences), and the distribution of the cell cycle was analyzed by flow cytometry using the Guava easyCyte 6HT-2L (Merck KGaA) (19). The results were analyzed using ModFit LT™ software (Windows version 4.0; Verity Software House).

The migratory and invasive abilities of PCa cells were investigated using a Transwell chamber (Corning, Inc.). For invasion assays, Matrigel (BD Biosciences) was thawed at 4°C overnight and diluted in serum-free medium. Then, 50 µl diluted Matrigel was added to the upper chamber and incubated at 37°C for 20 min. Cells (1×10⁵) were seeded into the upper chamber with serum-free medium containing 0.5% BSA, while the lower chamber was filled with 600 µl medium containing 10% FBS. After incubation for 24 h at 37°C, the cells were fixed with 4% polyoxymethylene for 15 min at room temperature, and then stained using the Hematoxylin and Eosin Staining Kit (Beyotime Institute of Biotechnology). The cells that had not passed through the polycarbonate membrane were removed with a cotton swab, and the invasive cells were visualized using a Leica DM500 microscope and counted in five randomly selected fields (magnification, ×100). For migration assays, all conditions were consistent, except for the use of Matrigel (20).

Male BALB/c nude mice (n=12) were obtained from the Shanghai Laboratory Animal Center (CAS), and were used at 5 weeks of age. The mice were housed and maintained in specific pathogen-free conditions under a 12 h light-dark cycle at 25°C, with free access to food and water. The tumor xenograft experiments were approved and conducted according to the guidelines provided by the Experimental Animal Center of Zhejiang Chinese Medical University (no. SYXK-2018-0012). PC3 cells with or without ZFP403-knockdown were harvested, resuspended in serum-free DMEM medium (2×10⁶) and subcutaneously injected into the dorsa of the mice (21). Animal health and behavior were monitored daily. When the tumor burden had reached ~50 mm³, the sizes were measured every 3 days and the tumor volume was calculated using the following formula: V=1/2 (length × width²). After 40 days, the mice were sacrificed by cervical dislocation. Death was verified by the absence of a heartbeat and the onset of rigor mortis.

The data are presented as the mean ± SD. Comparison between multiple groups was performed using one-way ANOVA followed by Tukey's post hoc test, while a paired Student's t-test was used for comparisons between 2 groups. All data analyses were performed using GraphPad Prism version 5 (GraphPad Software, Inc.), and P<0.05 was considered to indicate a statistically significant difference.

Data from the GEPIA2) database were used to determine the association between ZFP403 and PCa. Differential gene expression analysis revealed that ZFP403 expression was lower in PCa tissues than in normal tissues (Fig. 1A). To further examine the expression level of ZFP403 in PCa, 19 groups of PCa and corresponding adjacent tissues were analyzed using IHC. The results revealed that the expression of ZFP403 in PCa tissues was significantly lower than that in adjacent normal tissues (P<0.01; Fig. 1B and C). RT-qPCR and western blot analysis were then used to detect the ZFP403 level in normal prostatic epithelial cells (RWPE-1) and PCa cell lines (LNCaP, PC3, DU145 and 22RV1). As shown in Fig. 1D and E, the expression levels of ZFP403 were lower in cancer cells than in RWPE-1 cells (P<0.01). These data indicate that ZFP403 may function as a tumor suppressor in PCa.

To evaluate the role of ZFP403 in the proliferation PCa cells, human PCa cell lines (PC3 and DU145), which are insensitive to androgens, were selected for transfection with ZFP403-shRNA lentivirus. The results revealed that the expression of ZFP403 was markedly decreased in the ZFP403-knockdown cells compared with that in the negative control (NC) cells, though to greater degree in cells transfected with ZFP403-shRNA-1 (Fig. 2A and B). Plate colony formation assays were performed to determine the effects of ZFP403 on colony formation. The results demonstrated that knocking down ZFP403 significantly increased PC3 and DU145 cell colony numbers (Fig. 2C). Furthermore, in the soft-agar colony formation assay, ZFP403-knockdown resulted in a marked increase in the number of colonies (Fig. 2D), indicating that ZFP403 inhibits tumorigenicity in vitro.

In order to further examine the effect of ZFP403 on the proliferation of PCa cells, the cell cycle distribution was detected by flow cytometry. The results revealed that the number of cells transitioning from the S phase to the G₂/M phase was significantly increased in PCa cells in which ZFP403 was knocked down (Fig. 3A and B). On this basis, western blot analysis was performed to detect the expression of G₂/M phase-related proteins. The results demonstrated that knocking down ZFP403 upregulated the expression of cyclin B1, cdc2 and cdc25C (Fig. 3C). These data indicate that silencing ZFP403 retains the viability and promotes the proliferation of PCa cells.

To determine the potential role of ZFP403 in the metastasis of PCa, the effects of ZPF403 on migration and invasion were evaluated using Transwell chamber migration and invasion assays. The results revealed that the cell migratory and invasive abilities were significantly promoted following ZFP403-knockdown (Fig. 4A and B).

Further experiments demonstrated that ZFP403-knockdown in PC3 and DU145 cells resulted in downregulation of the epithelial cell marker E-cadherin, and the upregulation of the interstitial cell marker vimentin. In addition, β-catenin, heparanase and matrix metalloproteinase 2 (MMP2) were also upregulated, as shown in Fig. 4C. Taken together, these results suggest that silencing ZFP403 promotes PCa metastasis by enhancing cell migration and invasiveness.

In order to examine the effect of ZFP403 on tumor development and progression in vivo, a tumor xenograft model of PCa cells was established by subcutaneous inoculation of BALB/c nude mice. Body weight and tumor volume were measured every 3 days for 40 days. The growth rate of subcutaneous tumors in the ZFP403-knockdown group was significantly higher than that in the NC group. However, no significant difference in body weight was observed between the groups (Fig. 5A-D). In addition, compared with the NC group, the mRNA and protein levels of ZFP403 were decreased in the ZFP403-knockdown group, as shown by RT-qPCR and western blot analysis, respectively (Fig. 5E-F). These data suggest that decreased ZFP403 expression is closely associated with the tumorigenicity and development of PCa in vivo.