The cell lines, RWPE-1 (cat. no. CRL-11609), LnCap (cat. no. CRL-1740), 22RV1 (cat. no. CRL-2505), DU145 (cat. no. HTB-81) and PC3 (cat. no. CRL-1435) were purchased from the American Type Culture Collection. RWPE-1 cells were maintained in keratinocyte serum-free medium (Thermo Fisher Scientific, Inc.), DU145 cells were maintained in DMEM (Thermo Fisher Scientific, Inc.), and LnCap, 22RV1 and PC3 cells were maintained in RPMI 1640 (Sigma-Aldrich; Merck KGaA). The media were supplemented with 10% FBS (Thermo Fisher Scientific, Inc.), and the cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂. DU145 and PC3 cells were transfected with negative control (NC) small interfering (si)RNA and ADAR1-specific siRNAs (Shanghai Gene Pharma Co., Ltd.), and the suppressed expression of ADAR1 was confirmed using western blot analysis.

Preliminary screening of ADAR1 protein expression via immunohistochemistry (IHC) and western blot analysis with an antibody against ADAR1, was conducted on tissues from patients diagnosed with benign prostatic hyperplasia (BPH), PCa and CRPCa at The Fifth Affiliated Hospital of Guangzhou Medical University.

All procedures carried out in studies involving patients were conducted in accordance with the ethical standards of the institutional and/or national research committee, as well as with the 1964 Declaration of Helsinki and its later amendments, or comparable ethical standards. The present study was a retrospective study reviewed and approved by the Ethics Committee, The Fifth Affiliated Hospital, Guangzhou Medical University (Guangzhou, China). Written informed consent was obtained from all patients.

Samples were obtained from patients (28 male patients, including 10 BPH, 10 PCa and 8 CRPCa cases; age, 63–74 years) who underwent surgery at the Department of Urology, the Fifth Affiliated Hospital of Guangzhou Medical University (Guangzhou, China) between March 2019 and September 2020. BPH tissue samples were obtained from transurethral resection of the prostate; PCa and CRPCa tissue samples were obtained from radical prostatectomy. Pathological evaluation of these tissues was confirmed. Patients with CRPCa were selected according to the European Association of Urology guidelines (10). The inclusion criteria were as follows: i) Histologically confirmed diagnosis of BPH, PCa or CRPCa; ii) aged between 60 and 75 years; and iii) willing to donate tissue samples. Patients who had incomplete information or a history of other malignant tumors were excluded. In the present study, patient characteristics (age, prostate volume, preoperative prostate-specific antigen level, Gleason score and clinical stage) were collected retrospectively. The characteristics of the patients are displayed in Table I.

A standard IHC protocol was used to stain the tissue samples, using a rabbit monoclonal antibody against ADAR1 [Cell Signaling Technology, Inc.; cat. no. 81284] (11). Briefly, paraffin-embedded tissue sections were deparaffinized, rehydrated and subjected to antigen retrieval. Samples were incubated with anti-ADAR1 diluted with TBS (1:800). All samples were observed under a light microscope (Carl Zeiss AG) and automated image quantification was conducted using ImageJ2× software (National Institutes of Health).

The tissue and cell samples were homogenized in lysis buffer containing: 0.1 mol/l NaCl, 0.01 M Tris-HCl (pH 7.5), 1 mM EDTA and 1 µg/ml aprotinin. After centrifugation at 16,000 × g for 5 min (4°C), total protein content in the supernatants was determined using a Bradford Protein Assay kit (Bio-Rad Laboratories, Inc,). In total, 40–80 µg protein was separated via 8% SDS-PAGE and then transferred to nitrocellulose membranes (12,13). The membranes were blocked for 60 min with blocking buffer [composed of 5% skimmed milk in TBS-Tween-20 (0.25 M Tris-HCl pH 7.5, 0.1% Tween-20 and 0.15 M NaCl)], at 37°C, and subsequently incubated with anti-GAPDH (1:1,000; cat. no. 5174), anti-β-actin (1:1,000; cat. no. 4970), anti-β-tubulin (1:1,000; cat. no. 2128), anti-phosphorylated (p)-H2A.X (1:2,000; cat. no. 2577), anti-H2A.X (1:2,000; cat. no. 7631), anti-cleaved caspase-3 (1:1,000; cat. no. 9661), anti-caspase-3 (1:1,000; CST; cat. no. 9662), anti-cleaved PARP (1:500; cat. no. 5625) and anti-PARP (1:500; cat. no. 9542) antibodies (all CST Biological Reagents Co., Ltd.) overnight at 4°C. The primary antibodies were detected using an HRP-conjugated secondary anti-rabbit IgG antibody (1:2,000; cat. no. 7076; CST Biological Reagents Co., Ltd.) for 1 h at 37°C. Bands were visualized using 3,3′-diaminobenzidine reagent at room temperature for 2 min, and were semi-quantified via scanning with a Molecular Imager (Bio-Rad Laboratories, Inc.). The results were analyzed with ImageJ2× and Origin 8.0 software (National Institutes of Health).

Total RNA was extracted from cell lines using the RNeasy mini kit (Qiagen, Inc.) per the manufacturer's protocol, and was quantified through spectrophotometric measurement using a NanoDrop system (Thermo Fisher Scientific, Inc.). qPCR analysis was conducted using the Verso One-Step SYBR qRT-PCR kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Primers were custom-synthesized and obtained from Guangzhou HYY Co. (http://www.hyymed.com/) (Table II). The amplification reaction was performed as follows: Initial denaturation for 5 min at 95°C, followed by 30 cycles at 95°C for 10 sec, 60°C for 15 sec and 72°C for 20 sec. β-actin was used as a control for relative quantification. Rotor-Gene Software (Qiagen, Inc.) was used for comparative concentration analysis (14,15). All reactions were carried out in triplicate, and the 2^−ΔΔCq method was used to determine the relative quantification of mRNA expression (16).

DU145 and PC3 cells were seeded into 6-well plates at a density of 1×10⁴ cells/well, and transfected with negative control siRNA (NC-siRNA sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′) or ADAR1-specific siRNA (ADAR1-siRNA-a sense, 5′-GCAUCUGACCCGUGCUAUUTT-3′ and antisense, 5′-AAUAGCACGGGUCAGAUGCTT-3′; ADAR1-siRNA-b sense, 5′-GCAGCCCAUUUAUCUCAAATT-3′ and antisense, 5′-UUUGAGAUAAAUGGGCUGCTT-3′; and ADAR1-siRNA-c sense, 5′-GCUUCAACACUCUGACUAATT-3′ and antisense, 5′-UUAGUCAGAGUGUUGAAGCTT-3′) (final concentration, 20 nmol/l; all Shanghai GenePharma Co., Ltd.) using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following culture for 48 h at 37°C, transfection efficiency was assessed by western blotting. After transfection, cells were cultured for 24 h and then used for subsequent experimentation.

Cell viability was measured using the CCK-8 kit (Dojindo Molecular Laboratories, Inc.) following the manufacturer's protocol. Briefly, cells were seeded into a 96-well plate at a density of 8×10³ cells/well, and incubated with CCK-8 reagent at 37°C for 4, 24, 48 and 72 h. The optical density was measured at 450 nm to estimate the number of viable cells.

Apoptosis was analyzed using an Annexin V-FITC/PI kit (eBioscience; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Cells in each group were seeded into a 6-well plate at a density of 1×10⁵ cells/well, and transfected with NC siRNA and ADAR1-specific siRNAs. After incubation for a further 2 days, the cells were harvested, washed with PBS and resuspended in staining buffer (Annexin V-FITC/PI kit) at a final density of 3×10⁵/ml. Then, apoptosis was analyzed using a FACScan flow cytometer (FACSCalibur; BD Biosciences).

Statistical analysis was performed using SPSS software ver. 28.0 (SPSS, Inc.) and graphs were generated using GraphPad Prism 8 (GraphPad Software, Inc.). Statistical results are presented as the mean ± SD of ≥3 independent experiments. Unpaired Student's t-test was used to compare differences between two groups. One-way or two-way ANOVA were used for multiple comparisons, followed by Dunnett's or Tukey's post hoc tests. P<0.05 was considered to indicate a statistically significant difference.

Patient characteristics including age, prostate volume, preoperative prostate-specific antigen (PSA), source of tissue, Gleason score and clinical stage are summarized in Table I. In the present study, 28 male patients aged 63–74 were enrolled, including 10 BPH, 10 PCa and 8 CRPCa cases. The mean prostate volume in each group was 78.2 (65–91), 46.2 (37–53) and 46.3 (38–55) cm³, respectively. The mean preoperative PSA was 3.6 (0.8–10.5), 22.1 (12.6–32.1) and 57.6 (42.5–81.4) ng/ml, respectively. Histopathological Gleason score was determined based on the criteria of the World Health Organization. The mean Gleason score of patients with PCa was 7.8 (6–10), and that of patients with CRPCa was 8.8 (7–10). Tumors were staged according to the eighth edition American Joint Committee on Cancer Staging Manual (17). The number of PCa patients at stages T1, T2 and T3 was 3, 4 and 3, respectively. The number of CRPCa patients at stages T1, T2, T3 and T4 was 1, 3, 2 and 2, respectively.

The expression level of ADAR1 was determined using IHC images of PCa tissues. Via IHC staining and automated image quantification, ADAR1 expression was found to be significantly increased in PCa, compared with BPH tissues (Fig. 1A and B). Furthermore, PCa and CRPCa tissues were extracted and probed with an ADAR1 monoclonal antibody. Notably, higher protein expression levels of ADAR1 were detected in CRPCa tissues compared with PCa tissues (Fig. 1C and D). Furthermore, RT-qPCR and western blot analysis demonstrated that the mRNA and protein expression levels of ADAR1 were decreased in LNCaP cells and significantly increased in the CRPCa cell lines (22RV1, DU145 and PC3) compared with RWPE-1 cells (Fig. 1E-G). These results suggest that ADAR1 is upregulated in CRPCa tissues and cell lines.

DU145 and PC3 cells that are considered to be androgen receptor negative (with high expression levels of ADAR1) were transiently transfected with ADAR1-specific siRNAs. Total protein was isolated and analyzed via western blotting 48 h post-transfection. Compared with the blank (no siRNA) and mock-transfected cells (DU145 si-NC and PC3 si-NC), the expression level of ADAR1 was markedly suppressed in cells transfected with ADAR1 siRNAs (Fig. 2A-D).

Next, the potential effects of ADAR1-knockdown on the proliferation of DU145 and PC3 cells were assessed. As shown in Fig. 2E and F, in both cell lines, ADAR1-knockdown significantly inhibited cellular proliferation compared with the untransfected cells after 24 and 48 h (P<0.05). However, there was no statistically significant difference in cellular proliferation 72 h post-transfection.

Flow cytometry was used to assess whether ADAR1-knockdown was associated with DU145 and PC3 cell apoptosis. The results demonstrated that the apoptotic rate of ADAR1 siRNA-transfected cells was increased compared with that of the untransfected cells (Fig. 2G-J). Thus, these results indicated that knockdown of ADAR1 expression inhibited the proliferation and induces the apoptosis of DU145 and PC3 cells.

Next, it was determined whether the phosphorylation of H2AX during ADAR1-knockdown induced the apoptosis of DU145 and PC3 cells. The expression levels of p-H2A.X, H2A.X, cleaved caspase-3, caspase-3 cleaved PARP and PARP were detected using western blotting (Fig. 3A and B). Results showed that DU145 si-ADAR1 cells and PC3 si-ADAR1-c cells exhibited markedly increased activation of caspase-3 and cleavage of PARP, which was accompanied by the phosphorylation of H2AX (Fig. 3C and D). Collectively, these data confirm that ADAR1-knockdown inhibits the proliferation and induces the apoptosis of DU145 and PC3 cells by promoting the phosphorylation of H2A.X.