Identification of endonuclease domain-containing 1 as a novel tumor suppressor in prostate cancer

We examined a total of 30 BPH samples and 50 PCa samples in this study. The diagnosis was confirmed histologically after surgery. The BPH tissues and PCa tissues were respectively from patients treated by transurethral resection of prostate and laparoscopic radical prostatectomy in the Third Affiliated Hospital of Sun Yat-sen University during 1st January 2012 and 31st December 2015. IHC was performed as described previously [18]. Briefly, tissues were deparaffinized and then antigen retrieval was done in 10 mmol/L citrate buffer (pH 6.0) for 15 min at 100 °C. After blocking endogenous peroxidase with methanol containing 0.3% hydrogen peroxide (H₂O₂) for 15 mins, tissue sections were incubated overnight at 4 °C using a rabbit polyclonal ENDOD1 antibody (Abcam, ab121293, 1:500). All immunostained sections were reviewed by two pathologists independently who were blinded to the patients’ clinicopathological information. The analysis of immunostaining intensity was acquired by multiplying the level of staining intensity (negative = 0, weak = 1, moderate = 2, strong = 3) and percentage of positively stained cells (0% = 0, <10% = 1, 11–50% = 2, 51–80% = 3, >80% = 4), as described previously [19]. This study was approved by our Institutional Ethics Committee and all patients had signed the informed consents for using their surgically-resected tissues and related information.

Human prostatic cancer cell lines DU145 (HTB-81™), PC3 (CRL-1435™), LNCaP (CRL-1740™) and 22Rv1 (CRL-2505™) and the non-malignant human prostatic normal epithelial cell RWPE-1 (CRL-11609™) were obtained from the American Type Culture Collection (ATCC) and routinely cultured in RPMI1640 medium (GIBCO, USA) supplemented with 10% fetal bovine serum (FBS) in a humidified 37 °C incubator containing 5% CO₂, according to the ATCC instructions.

Total RNA was extracted from RWPE-1, DU145, PC3, LNCaP and 22Rv1 using Trizol reagent (Invitrogen Life Technologies, CA, USA). cDNA was synthesized using 2 μg total RNA and SYBR® premix Ex Taq II (Takara, Japan). qPCR was performed on the ABI Prism 7500 fast Sequence Detection System (Applied Biosystem). The 2^-ΔΔCt method was performed to analyze relative quantities for the level of gene expression. The following primers were used: GAPDH forward 5′-TCCTCTGACTTCAACAGCGACACC-3’and reverse 5′-TCTCTCTTCCTCTTGTGCTCTTGG-3′, ENDOD1 forward 5′-GACCGCATCCCCGTGTA-3′ and reverse 5′-AATCGCCTCCTCAAGGTT-3′.

The ENDOD1-expressing construct pCMV-N-Flag-ENDOD1 was performed as previously reported [20]. Genomic cDNA from PC3 cells was used as templates to amplify the upper and lower genes with the sites of EcoR I and Xho I restriction enzymes. Taq enzyme was used to obtain amplifications. After digested by the Xho I and EcoR I restriction enzymes, ENDOD1 and pCMV-N-Flag plasmid were extracted and purified using Gel Extraction Kit (Omega biotech) according to the manufacturer’s instructions. Then products were mixed, connected with DNA Ligation Kit (Takara) and transformed into DH5α peptide (Takara). After coated the lysogeny broth solid medium with kanamycin 100 μg/ml for 16 h, single colonies were chosen randomly. Colonies were inoculated in the lysogeny broth medium containing with kanamycin 100 μg/ml overnight at 37 °C, 150 r/min and then plasmid DNA was extracted according to instructions of Endo-Free Plasmid Midi Kit (Omega biotech). The plasmids were sequenced before transfection (BGI tech, China). Cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were also transfected with siRNA specific to ENDOD1 (5′-CAGAUUACCUUGAUUCUGATT-3′) (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen), following the manufacturer’s instructions. The concentration of siRNA used was 100 nM. After 6 h of transfection, the complete culture media was added. The assays were done 48 h post-transfection.

The DU145 with overexpression and negative control were cultured in 96-well culture plates at the number of 3000 cells per well, in RPMI-1640 medium containing 10% FBS. LNCaP transfected with siRNA and si-NC were cultured at a cell density of 5000 cells/well under the same conditions as DU145. The growth of the cells was analyzed by MTT assay (KeyGEN BioTECH, Nanjing, China). Optical density (OD), which was detected by an Automatic microplatereader (TEAN, Swiss) at a 490 nm wave length. The rate of cell proliferation was calculated. Three independent experiments were performed over multiple days.

Cell cycle and apoptosis studies were performed using FCM on DU145 transfected with ENDOD1 overexpression and the control plasmids, LNCaP transfected with siRNA and si-NC, respectively. Cells were seeded at a concentration of 3 × 10⁶ cells/mL. For cell cycle analysis, the cells were dispersed in 500 μL PBS (1%, pH 7) with PI (50 μg/mL) and RNase A (100 μg/mL) and kept in dark for 15 min before FCM analysis. For apoptosis study, cells were treated with PI (50 μg/mL), annexin V-FITC conjugate (5 μg/mL)(KeyGEN BioTECH, Nanjing, China) and dispersed in PBS (1%, pH 7). Each analysis was performed at least thrice (n = 3) and a count of a minimum of 10,000 events was taken for each analysis. Data are represented as mean ± S.D.

The migration and invasion activity of DU145 cells with overexpression ENDOD1 and LNCaP cells with siRNA were assessed using transwell migration assays and matrigel invasion assays as previously described [21]. Cells were seeded into the upper chamber of the transwell in 24-well plates (membrane pore size, 8 μm; Corning Incorporated; Corning, NY, USA) with Matrigel (BD Pharmingen), and 500 μl RPMI-1640 medium with 15% FBS was added into the lower chamber. After 24 h, the bottoms of the inserts were fixed in paraformaldehyde solution for 10 mins and stained with 0.1% crystal violet staining solution. The cells invading into the bottom-lower surfaces of inserts were measured by using microscope at ×100. Results were repeated in triplicate.

The western blot was performed as previously reported [22]. Cells were lysed by using RIPA buffer containing 1% PMSF. Protein concentration was quantified with the BCA Protein Kit (KeyGEN BioTECH, Nanjing, China) and equal amounts of proteins were loaded and separated by 12% SDS-PAGE, then transferred a to a PVDF membrane (Millpore, USA) and probed with primary antibody against ENDOD1 (Abcam, ab121293,1:2000) using antibodies against ENDOD1 (Abcam, ab121293, 1:2000). All the levels of protein were normalized to GAPDH (Abcam, ab9485,1:2500).

We detected ENDOD1 protein expression in 30 BPH patients and 50 PCa patients by IHC. The mean ages of BPH patients and PCa patients were 54.2 (45–84) years and 68.3 (50–82) years, respectively. The average PSA value was 9.4 (2.1–25.6) ng/mL in BPH patients and 24.2 (3.8–175.5) ng/mL in PCa patients. The basic clinical characteristics of PCa patients are summarized in Table 1. No association was found between ENDOD1 expression and age (P = 0.749), preoperative PSA (P = 0.196), clinical T stage (P = 0.443), biopsy Gleason score (P = 0.191), pathological T stage (P = 0.292), seminal vesicle invasion (P = 0.139), lymphnode involvement (P = 0.064). While a significant difference was revealed between ENDOD1 expression and postoperative Gleason score (P = 0.011) and surgical margin status (P = 0.024). Representative images of immunostaining in BPH tissues and PCa tissues are shown in Fig. 1a-f. Immunoreactivity score analysis demonstrated that there was no difference in ENDOD1 expression between BPH samples and PCa tissues with low Gleason score (Gleason score < 7) (Fig. 1g). However, PCa tissues with high Gleason score (Gleason score ≥ 7) displayed significantly lower ENDOD1 expression than that with low Gleason score and BPH (Fig. 1g). These findings suggested that ENDOD1 down regulation might play a role in the progression of PCa.

In order to investigate the expression of ENDOD1 in PCa cells, Real-time PCR analysis of ENDOD1 mRNA expression was performed in a panel of normal prostatic epithelial and PCa cell lines (22Rv1, LNCaP, PC3 and DU145). The data indicated that the level of ENDOD1 expression was markedly increased in LNCaP and 22Rv1 cells lines (mean ± SD, n = 3, P < 0.05) and decreased in PC3 and DU145 (mean ± SD, n = 3, P < 0.05) compared to the normal epithelial cell RWPE1 (Fig. 2a). Western blot showed that androgen-sensitive LNCaP cells had the highest protein expression level of ENDOD1, whereas castration resistant PCa cell lines PC3 and DU145 had significantly lower levels of ENDOD1 (Fig. 2b and c). However, the protein level is not proportional to the mRNA level in 22RV1 (Fig. 2b and c). These results indicated that ENDOD1 may be involved in the progression from androgen dependence (AD) to androgen independence (AI). For further investigation on the impact of ENDOD1 in tumor biology of PCa, we selected LNCaP and DU145 cells as study models.

To explore the effect of ENDOD1 on proliferation of PCa cells, eukaryotic overexpression plasmid pCMV-N-Flag-ENDOD1 and specific small siRNA was transfected into DU145 and LNCaP cells respectively. The expression of ENDOD1 was confirmed by western blot (Fig. 3a and b). We next investigated the cell proliferation rate of these cells by MTT assay. After transfection with ENDOD1, DU145 cells showed significantly decreased OD values in 96 h post-transfection, suggesting an inhibitory effect of ENDOD1 on proliferation of DU145 (P < 0.05, Fig. 3c). Conversely, LNCaP cells proliferated remarkably faster after siRNA-mediated down regulation of ENDOD1 (P < 0.05, Fig. 3d). Therefore, ENDOD1 suppresses cell proliferation in PCa cells.

To identify changes in cell cycle distribution, we performed FCM after manipulation of ENDOD1 expression. Overexpression of ENDOD1 in DU145 cells led to notable increases in G0/G1 phase arrest, with a corresponding decrease in the percentage of cells in the G2/M and S phases, compared with control group (Fig. 4a and b, P <0.01). LNCaP cells transfected with siRNA reversed the effect, with a remarkable increase in the percentage of cells in the G2/M phase (Fig. 4c and d, P <0.01). Moreover, we measured cell apoptotic rate to determine whether ENDOD1 promotes apoptosis. As shown in Fig. 4e and f, the percentage of apoptotic cells were 3.28% vs 1.02% for DU145 cells with ENDOD1 expression and the control, respectively and were 5.53% vs 5.66% for LNCaP cells with siRNA and si-NC, respectively, showing no statistical difference. Collectively, these data suggest that ENDOD1 inhibits cell cycle progression at the G0/G1 arrest and has no effect on cell apoptosis.

As for the role of ENDOD1 in migration and invasion of PCa cells, we performed transwell migration and invasion assays. ENDOD1 overexpression significantly suppressed cell migration and invasion in DU145 (Fig. 5a and c) whereas knockdown of ENDOD1 by siRNA promoted cell migration and invasion in LNCaP cells (Fig. 5b and d). Taken together, there is an obvious negative regulation of migration and invasion by ENDOD1 expression in PCa cells.