The nHDPCs were purchased from Innoprot (Biscay, Spain) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Life Technologies, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO₂.

The viability of the nHDPCs was measured using a water-soluble tetrazolium salt (WST-1) assay (EZ-Cytox Cell Viability Assay kit; Itsbio, Seoul, Korea). For the cell viability assay, the nHDPCs were plated at a density of 5×10³ cells/well in 96-well plates. After 24 h, the cells were treated with doses of DHT between 0 and 1 mM at 37°C for 24, 48, or 72 h. The cells were then incubated with WST-1 reagent at 37°C for 30 min, and the optical density was determined at 450 nm using a microplate reader (iMark; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

A propidium iodide (PI) staining based cell cycle assay was performed using standard procedures, as described previously (10). The nHDPCs (2×10⁶) were plated in 60 mm culture dishes and treated with DHT for 24 h. The cells were then trypsinized with 0.25% Trypsin-EDTA (Gibco Life Technologies) at 37°C, pelleted, washed with phosphate-buffered saline (PBS), and fixed with 70% ethanol at 4°C for 3 h. The DNA in the fixed cells was stained using staining solution containing 50 μg/ml PI (Sigma-Aldrich), 0.5% Triton X-100 (Bioshop, Burlington, ON, Canada), and 100 μg/ml RNase (Bioshop) at 37°C for 1 h. Following staining, the cells were analyzed using a FL2 channel with an excitation wavelength of 488 nm and an emission wavelength of 578 nm, on a FACSCaliber flow cytometer (BD Biosciences, San Jose, CA, USA).

The measurement of ROS was performed, as previously reported, using 2′,7′-dichlorofluorescein diacetate (DCF-DA) (19). The nHDPCs (2×10⁶) were plated in 60 mm culture dishes and treated with DHT at 37°C for 24 h. 2′, 7′-Dichlorodihydrofluorescin diacetate (DCF-DA; 20 μM) was added to the culture medium, and the cells were incubated at 37°C for 1 h. The cells were then trypsinized with 0.25% Trypsin-EDTA at 37°C, pelleted, washed with PBS, and analyzed using a FL1 channel with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACSCaliber flow cytometer (BD Biosciences).

For the detection of senescent cells, an SA-β-gal assay was performed, as previously described (20). Briefly, the nHDPCs (2×10⁶) were plated in 60 mm culture dishes and treated with DHT at 37°C for 24 h. The cells were then fixed with Fixative Solution (Senescence Detection kit; Biovision, Milpitas, CA, USA) and stained using a Staining Solution mix (Senescence Detection kit) supplemented with X-gal at 37°C for 24 h. Images of the SA-β-gal stained cells were captured using a camera mounted to a light microscope (CKX41; Olympus Corporation, Tokyo, Japan), and the number of stained cells were counted in five randomly selected microscopic fields from each condition.

The RNA in the cells was isolated using TRIzol reagent (Gibco Life Technologies), according to the manufacturer’s instructions. The RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and the RNA quality was evaluated using spectrophotometry at the 260/280 nm ratio (Ultrospec 2100 Pro UV-Vis; Amersham Biosciences, GE Healthcare Life Sciences, Piscataway, NJ, USA). Samples with an RNA integrity score >7.8 and an RNA quality score >2.0 were used for the microarray. A total of 100 ng RNA was labeled with cyanine dye (Cy3) using an Agilent miRNA labeling kit (Agilent Technologies). The labeled RNAs were purified using Micro Bio-Spin P-6 columns (Bio-Rad Laboratories, Inc.) and hybridized using a SurePrint G3 Human v16 miRNA Microarray kit (8×60 K; Release 16.0; Agilent Technologies) at 65°C for 20 h. The microarray was scanned using an Agilent microarray scanner (Agilent Technologies), and the images were analyzed using Agilent Feature Extraction version 10.7 software (Agilent Technologies). The digitized data were analyzed and the fold change was determined using GeneSpring GX version 11.5 software (Agilent Technologies).

The putative target genes of significant miRNAs were identified using the probability of interaction by target accessibility (PITA; http://genie.weizmann.ac.il), microRNAorg (http://www.microrna.org) and TargetScan (http://www.targetscan.org) target prediction systems. The Gene Ontologies (GOs) of the putative target genes were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resource 6.7 (http://david.abcc.ncifcrf.gov).

The data are presented as the mean ± standard deviation. Statistical significance was calculated using Student’s two-tailed t-test. Statistical analyses were conducted using Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA). P<0.01 was considered to indicate a statistically significant difference, unless otherwise indicated.

To determine whether DHT was associated with cell viability in nHDPCs, the present study analyzed the viability of DHT-treated nHDPCs after 24, 48, and 72 h using a WST-1 assay. Low concentrations of DHT (<0.1 mM) demonstrated no significant toxicity in the nHDPCs at any of the time-points assessed. However, as shown in Fig. 1, cytotoxicity was significantly increased by 1 mM DHT in the nHDPCs at every time-point assessed. Thus, it was determined that 1 mM DHT-induced cytotoxicity in the nHDPCs following exposure for ≥24 h, which led to an exposure duration of 24 h being selected for use in the subsequent experiments.

Previous experiments established that high levels of DHT induce apoptosis (14,21). In agreement with the previous experiments (Fig. 2), the present study demonstrated that 1 mM DHT increased cell death between 3.36 and 15.62% in the nHDPCs. In addition, the G1/G2 ratio was significantly increased by concentrations of DHT >10⁻⁶ M, in a dose-dependent manner. The DHT-induced increment in G1/G2 ratio indicated that DHT-induced G2 cell cycle arrest. Therefore, high-doses of DHT reduced cell viability through induction of cell death and G2 cell cycle arrest in the nHDPCs.

DHT can induce ROS in prostate cancer cell lines, which express the androgen receptor at a high level (14,22–24). Additionally, ROS are a key inducer of retinoblastoma-mediated senescence (25). As nHPDCs also express androgens at a high level (26), the present study investigated whether 1 mM DHT-induced ROS in these cells. The levels of ROS were determined using DCF-DA staining in untreated nHDPCs and in 1 mM DHT-treated nHDPCs. As shown Fig. 3A, DHT significantly increased the level of ROS in the nHDPCs. In addition, the cellular effect underlying the effect of 1 mM DHT in enhancing ROS levels in the nHDPCs was investigated. As shown in previous experiments in a prostate cell line (23), accumulated ROS induced senescence in the nHDPCs, as assessed by SA-β-gal activity (Fig. 3B).

As DHT induced growth arrest, cell death, cell cycle arrest, ROS production and senescence, comparative microarray analysis of miRNAs was performed to identify the miRNA signatures in the DHT-treated nHDPCs. Total RNA was extracted from the untreated nHDPCs and nHDPCs treated with 1 mM DHT for 24 h. The total RNA was labeled with Cy3 and hybridized to microarray-containing probes for 1,205 annotated miRNAs. The untreated cells were then compared with the 1 mM DHT-treated nHDPCs, in which 55 miRNAs that were upregulated and 6 were downregulated, by more than two-fold (Table I). Among the five miRNAs significantly upregulated in the DHT-treated nHDPCs, the level of miR-3663-3p increased by 219.04-fold, miR-485-3p by 200.81-fold, miR-7 by 173.64-fold, miR-125a-3p by 154.55-fold, and miR-4271-by 108-fold. In addition, in the five miRNAs, which were significantly downregulated in the DHT-treated nHDPCs, the level of miR-450a decreased by 95.69-fold, miR-1181 by 93.76-fold, miR-3656 by 2.84-fold, miR-4286 by 2.29-fold and miR-370 by 2.24-fold.

Subsequently, the putative target genes of DHT-regulated miRNAs were identified using the PITA, microRNAorg and Targetscan target prediction systems (Table II). A total of 587 putative target genes of the upregulated miRNAs and 140 putative target genes of the downregulated miRNAs were identified in PITA. Using microRNAorg, 488 putative target genes of upregulated miRNAs and 312 putative target genes of downregulated miRNAs were found, and 691 putative target genes of upregulated miRNAs and 219 putative target genes of down regulated miRNAs were identified using Targetscan. Of these, 339 were overlapping target genes of upregulated miRNAs and 111 were overlapping target genes of downregulated miRNAs in all three target prediction systems.

To investigate a association between the aforementioned effects of DHT and the putative miRNA target genes, GO analysis of each putative target gene was performed using DAVID. The genes were classified according to GO terms associated with the five effects of DHT and the number of putative target genes associated with each GO term were counted. As shown in Table III, the putative target genes of the uppregulated and downregulated miRNAs were associated with five antioxidant-associated GO terms, 17 apoptosis and cell death-associated terms, 11 proliferation and cell growth-associated terms, 1 age associated term and 14 cell cycle-associated GO terms. The miRNAs and their putative target genes are shown in Table IV. Overall, these results demonstrated that DHT exerted negative effects, which were associated with an alteration in cellular miRNA expression profiles.