The Prostate-Associated Gene 4 (PAGE4) Could Play a Role in the Development of Benign Prostatic Hyperplasia under Oxidative Stress

Benign prostatic hyperplasia (BPH) is a common disease in elderly men with uncertain molecular mechanism, and oxidative stress (OS) has also been found associated with BPH development. Recently, we found that prostate-associated gene 4 (PAGE4) was one of the most significantly changed differentially expressed genes (DEGs) in BPH, which can protect cells against stress stimulation. However, the exact role of PAGE4 in BPH remains unclear. This study is aimed at exploring the effect of PAGE4 in BPH under OS. Human prostate tissues and cultured WPMY-1 and PrPF cells were utilized. The expression and localization of PAGE4 were determined with qRT-PCR, Western blotting, and immunofluorescence staining. OS cell models induced with H2O2 were treated with PAGE4 silencing or PAGE4 overexpression or inhibitor (N-acetyl-L-cysteine (NAC)) of OS. The proliferation activity, apoptosis, OS markers, and MAPK signaling pathways were detected by CCK-8 assay, flow cytometry analysis, and Western blotting. PAGE4 was shown to be upregulated in human hyperplastic prostate and mainly located in the stroma. Acute OS induced with H2O2 increased PAGE4 expression (which was prevented by OS inhibitor), apoptosis, cell cycle arrest, and reactive oxygen species (ROS) accumulation in WPMY-1 and PrPF cells. siPAGE4 plus H2O2 potentiated H2O2 effect via reducing the p-ERK1/2 level and increasing p-JNK1/2 level. Consistently, overexpression of PAGE4 offset the effect of H2O2 and partially reversed the PAGE4 silencing effect. However, knocking down and overexpression of PAGE4 alone determined no significant effects. Our novel data demonstrated that augmented PAGE4 promotes cell survival by activating p-ERK1/2 and decreases cell apoptosis by inhibiting p-JNK1/2 under the OS, which could contribute to the development of BPH.


Introduction
Benign prostatic hyperplasia (BPH) refers to the nonmalignant growth of prostate tissue, and the incidence of this disease increases with age [1,2]. In fact, the histological prevalence of BPH is 50% to 60% in men at 60 years of age and 80% to 90% in men over 70 years of age [3]. The secondary lower urinary tract symptoms (LUTS) impair the quality of their life [4]. Histopathologically, it results from a loss of homeostasis between cell proliferation and cell death with cell proliferation being dominant [5]. In addition to androgen and age dependence, androgen-estrogen ratio, the interaction between stromal and epithelial cells, and inflammation, as well as growth factors, are the other accepted predisposing BPH factors [6][7][8]. However, the exact pathophysiology of these mechanisms remains unclear.
In the past decades, oxidative stress (OS) has been found associated with BPH development, progression, and the response to therapy [9]. OS is defined as the imbalance between the production and scavenging of reactive oxygen species (ROS) [10]. It is thought that OS could produce oxidative DNA damage, defective DNA repair, and genomic instability, leading to an increase in mutations, some of which may promote cell transformation and accelerate proliferation [11].
Genomics technologies have led to deeper understanding of many human diseases. More than 15 years ago, DNA microarrays were used to explore BPH [12]. Recently, our group had performed mRNA expression profiling of three normal human prostates and five BPH tissues, and our microarray data (serial number: GSE119195) identified a total of 198 differentially expressed genes (DEGs) [13,14]. Among these DEGs, prostate-associated gene 4 (PAGE4) was one of the genes significantly upregulated in BPH tissues. Although PAGE4 is expressed at basal levels in certain fetal and adult reproductive organs such as placenta and uterus [15], PAGE4 is clearly prostate-specific in men. In addition, its expression is highly dynamic. PAGE4 protein is highly upregulated in both fetal prostate epithelium and stromal cells at 21 weeks of gestational age [16], while it is almost undetectable in normal adult prostate [17]. Accumulated evidences have shown that PAGE4 is reexpressed in pathological human prostate tissues, especially in symptomatic benign prostatic hyperplasia [18], as well as prostate cancer [19][20][21]. PAGE4, as a kind of intrinsically disordered protein, can protect cells against stress stimulation [19]. In the cellular process, it has been found that PAGE4 is mainly located in mitochondria, which are the main site of oxidative stress. Indeed, studies found that PAGE4 was upregulated when prostate cancer cells were exposed to various stress inducers (including inflammatory stress and oxidative stress) [22]. However, how PAGE4 influences BPH under the condition of oxidative stress is still unclear.
In our current study, the relative expression of PAGE4 at transcriptional and translational levels was analyzed in human BPH prostates and human normal prostates. Furthermore, two cell models of PAGE4 deficiency or upregulation were established to investigate the functional activities of PAGE4. Also, the underlying mechanisms of PAGE4 protection of cells from OS were focused in the study.

Materials and Methods
2.1. Human Prostate Tissue Samples. Human hyperplastic prostate tissues were obtained from ten male patients (mean age, 68:3 ± 3:5 years) who underwent transurethral resection of the prostate in the Department of Urology, Zhongnan Hospital of Wuhan University. All samples exhibited BPH without tumor infiltration and intraepithelial neoplasia, which were confirmed by two separate pathologists. Normal prostate tissue was obtained from ten young brain-dead men (mean age, 31:7 ± 2:5 years) undergoing donation in the Organ Transplant Center of Zhongnan Hospital, with pathological exam showing no hyperplasia. Prostate tissues were divided into two strips and were, respectively, stored in liquid nitrogen for PCR and Western blotting analyses and in 10% neutral buffered formalin for immunofluorescence microscopy. All human samples were obtained after the approval of the Hospital Human Investigation Committee and receiving the written informed consent of all patients or their relatives. All human research was carried out in accordance with the principles of the Helsinki Declaration. The study was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University, and samples were col-lected and processed in accordance with the approved guidelines.

Cell
Culture and H 2 O 2 Treatment. SV40 large-T antigenimmortalized stromal cell line WPMY-1 (Cat. #GNHu36) was purchased from the Stem Cell Bank, Chinese Academy of Sciences in Shanghai, China. Cell lines were identified at the China Center for Type Culture Collection in Wuhan, China. Primary prostate fibroblast (PrPF) was purchased from the Procell Co., Ltd. In Wuhan, China.WPMY-1 cells were cultured in DMEM medium (Gibco, China) containing 1% penicillin G sodium/streptomycin sulfate and 5% fetal bovine serum (FBS; Gibco, Australia). Fibroblasts were cultured in a DMEM : F12 1 : 1 (Thermo Fisher Scientific, MA, USA) mixture supplemented with 10% FBS (Thermo Fisher Scientific, MA, USA), 1% penicillin-streptomycin, 1 μg/ml EGF (PeproTech, NJ, USA), and 1 μg/ml bFGF (PeproTech, NJ, USA). All the cell lines were cultured in a humidified atmosphere consisting of 95% air and 5% CO 2 at 37°C. Medium required by different cells were utilized to dilute 3% H 2 O 2 (BD Biosciences, USA) into concentrations of 100 μM, 200 μM, 400 μM, and 800 μM for subsequent experiments. To stimulate oxidative stress in vitro, medium containing 400 μM H 2 O 2 was incubated with the cells for 8 h prior to analysis. 5 mM N-acetyl-L-cysteine (NAC, Sigma-Aldrich) was given to cells for 8 h before collection.

CCK-8 Assay.
Cell Counting Kit-8 (MedChemExpress, China) was used to determine cell proliferation activity. After cell treatment, CCK-8 working solution of 10% volume fraction was added to the cells and cultured in a 37°C incubator for 4 h. The readings were recorded by a microplate reader (cat. no. SpectraMaxM2; Molecular Devices, Sunnyvale, CA, USA), and the absorbance at 450 nm was measured at the same time for each day.
2.4. Total RNA Extraction, Reverse Transcription, and Quantitative Real-Time PCR (qRT-PCR). Total RNA was extracted from prostate tissues and cells using the RNeasy Mini Kit (Qiagen, Germany) according to the instructions and a centrifuge (Eppendorf, USA) combined with QIAshredder (Qiagen, Germany) to increase the quantity and quality of total RNA isolated. Each RNA sample was digested with DNase I (RNase-Free DNase Set, Qiagen, Germany) to remove contamination of the genomic DNA. The quantity of isolated RNA was detected with a NanoDrop® ND-1000 UV-Vis spectrophotometer (Thermo Scientific, USA). 1 μg of total RNA isolated from prostate tissues or prostate cells was mixed with oligo (dT) 12-18 primers to synthesize first-strand cDNA by using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, China). 1 μg cDNA was used for each individual polymerase chain reaction (PCR) reaction in a final volume of 20 μl. All primers to be used with the SYBR Premix Ex Taq II (Takara Bio, China) were tested for optimal annealing temperatures, and PCR conditions were optimized with gradient PCRs on a Bio-Rad (Hercules, CA, USA) CFX96 system. The 2 -ΔΔct method was used to calculate the relative changes in expression.
. And the sense sequence of si-control was 5 ′ -UUCUCC GAACGUGUCAGUGACAUUAAGAUUCAGGGUTT-3′. After transfection by siPAGE4 for 48 h, alterations of PAGE4 at transcriptional and protein levels were evaluated by the qRT-PCR and Western blot. PAGE4 cDNA was polymerase chain reaction (PCR) amplified from a cDNA library of human prostate cell lines and then cloned into a 2x FlagpcDNA3 empty vector performed with a one-step method to construct the homologous recombination vectors. After transfection by plasmid for 48 h, alterations of PAGE4 at transcriptional and protein levels were evaluated by qRT-PCR and Western blot.
2.6. Western Blot Analyses. The prostate cells were sonicated and lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor (Sigma-Aldrich, USA) on ice for 30 min and then centrifuged at 12,000 g for 15 min to collect supernatant. The concentrations of protein were determined by Bradford protein assay (Bio-Rad, Germany) using bovine serum albumin (BSA) as standard. The isolated total protein was resolved using 6-15% SDS-PAGE and transferred to PVDF membrane (Millipore, USA). Membranes were blocked by 5% nonfat milk and incubated with primary antibodies (Table 2) at 4°C for overnight. After washing, the membranes were incubated with secondary antibody (Table 3) at room temperature for 2 h. Bands were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Scientific Fisher, Waltham, MA, USA) and detected by Kodak Biomax MR films.

Flow Cytometry Analysis.
For cell cycle analysis, 1 × 10 6 cells were harvested and fixed in 70% ice-cold ethanol at -20°C overnight. After centrifugation, pellets were resuspended with PBS containing 50 μg/ml propidium iodide (Sigma-Aldrich, USA) and 0.1 mg/ml RNase A (20 μg/ml in PBS) in the dark. After incubation at 37°C for 30 min, the DNA content distribution was analyzed by flow cytometry analysis (Beckman, Cat. #FC500). For apoptosis analysis, after transfection for 48 h, cells were collected, washed with PBS, and stained with FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, USA) and analyzed by the flow cytometry analysis.

ROS Detection by
Staining with DCFH-DA. The fluorescent probe 2 ′ ,7 ′ -dichlorofluorescin diacetate (DCFH-DA Sigma-Aldrich, USA) was used to evaluate intracellular ROS levels. After transfection of cells and growth for 48 h, 10 μmol of DCFH-DA (Sigma, USA) was added to 1 ml medium and incubated at 37°C for 30 min. Thereafter, the cells were washed three times with PBS and submitted to flow cytometry analysis.
2.9. Immunofluorescence Staining for Human Prostate Tissues. Frozen normal and BPH human prostate tissues were sectioned in 10 μm thick slices and thawed, mounted onto glass slides using a cryostat (Leica CM 1850, Wetzlar, Germany), air-dried, and fixed for 10 min in ice-cold acetone. Slides were washed in PBS and incubated for 2 h in a mixture of PBS supplemented with 0.2% Triton X-100 and 0.1% bovine serum albumin. After incubation overnight with the primary antibody mixture of α-SMA and PAGE4 antibody ( Table 2), the secondary antibodies (Jackson ImmunoResearch Inc.) labelled with FITC-conjugated antimouse IgG (1 : 200) and Cy3-conjugated anti-rabbit IgG (1 : 1000) were used to visualize the localization of the two primary antibodies. DAPI (Table 3) was used for staining the nucleus. Visualization was done with a Laser Scanning Confocal Microscope (Olympus, Tokyo, Japan).

Immunofluorescence Staining for Human Prostate Cells.
For cell immunofluorescence microscopy, WPMY-1 and PrPF cells were cultured as aforementioned, followed by seeding on 12 mm coverslips and washing by ice-cold phosphate-buffered saline (PBS, pH = 7:4). The coverslips were then fixed with 4% paraformaldehyde (PFA) for 30 min, followed by 0.1% Triton X-100 incubation and blocked in goat serum for 30 min at room temperature and then incubated with primary antibody ( Table 2) at room temperature for 2 h, washed with PBS, and incubated with Cy3-labeled or FITC-labeled secondary antibody (Table 3) for 1 h. Nuclei were labeled with DAPI (2 μg/ml). Visualization was done with a Laser Scanning Confocal Microscope (Olympus, Tokyo, Japan).
2.11. Statistical Analysis. All analyses were performed at least three times and represented data from three individual experiments. The data values are expressed as the means ± standard deviation (SD). Statistical analysis was performed using Student's t-test (two groups compared) and one-way ANOVA (multiple means compared) with SPSS 22.0. Statistical significance was considered as a p value < 0.05.

The Expression and Localization of PAGE4 in Human
Prostate Tissues. For human BPH and normal prostate samples, n = 10 each. In hyperplastic prostate, the mRNA level of PAGE4 was significantly increased over fifteenfold (Figure 1(a)), and the protein level of PAGE4 revealed significant upregulation, which could almost not be detected in normal prostate tissue (Figure 1(b)). Consistently, immunofluorescence staining demonstrated that PAGE4 was barely detectable in normal prostate tissue (Figure 1(c)) but it was richly expressed in hyperplastic tissues. Moreover, PAGE4 was mainly localized in the stroma (Figure 1(d)).       (Figure 3(a)). Therefore, si-PAGE4-1 and si-PAGE4-2 were selected for subsequent experiments, because they exhibited more than 75% inhibitory effect. Cell proliferation was further analyzed by CCK-8 assay for these transfected WPMY-1 and PrPF cells, which revealed that knockdown of PAGE4 significantly inhibited cell proliferation under OS. However, the downregulation of PAGE4 expression alone had no significant effect on cell proliferation (Figure 3(b)). In the presence of H 2 O 2 , the mRNA and protein levels of SOD1 and catalase (CAT) related to oxidative stress were significantly decreased (Figures 3(c)-3(e)). Parallelly, the accumulation of intracellular reactive oxygen species (ROS) was clearly elevated in aforementioned groups (Figures 3(f) and 3(g); Figure S1). However, silencing of PAGE4 alone had no effect on SOD1, CAT, and ROS.     Oxidative Medicine and Cellular Longevity in the GO/G1 phase by H 2 O 2 treatment, there was a corresponding shortening of the G2 phase, while there was no significant change in the S phase, and an increase of G0/G1 phase cells approximately 7.3%/6.5% and a decrease of G2 phase cells about 6.4%/5.3% in WPMY-1/PrPF were found. Moreover, the knockdown of PAGE4 not only enhanced the increase of H 2 O 2 -induced GO/G1 phase cells by about 6.6%/5.5% but also enhanced the decrease of H 2 O 2 -induced G2 phase cells by about 6.2%/5.4% in WPMY-1/PrPF (Figures 4(b) and 4(d)). However, siPAGE4 alone demonstrated no effect on cell apoptosis and cell cycle. Accordingly, significant changes were observed for cell apoptosisassociated proteins (BAX and Bcl-2) and cell cycle-related proteins (Cyclin D1 and CDK6) (Figure 4(e)). MAPK signaling pathways are known to regulate cell processes in various ways, including cell proliferation, differentiation, mitosis, and apoptosis. The phosphorylation levels of JNK1/2 and ERK1/2 in MAPK signaling pathways were determined via Western blot. The expression of phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated JNK1/2 (p-JNK1/2) was increased in the cells exposed to H 2 O 2 . The downregulation of PAGE4 attenuated H 2 O 2 -induced p-ERK1/2 activation, but enhanced p-JNK1/2 activation under OS ( Figure 5).

The Overexpression of PAGE4 Promotes the Cell Survival of Prostate Stromal Cells and Reduces ROS Accumulation under OS.
Additionally, we transfected the plasmid into WPMY-1 and PrPF cells to overexpress PAGE4. qRT-PCR revealed that the mRNA expression of PAGE4 was upregulated by about 3200/2825-fold in WPMY-1/PrPF ( Figure 6(a)). The protein level of PAGE4 was obviously increased (Figure 6(b)). Overexpression of PAGE4 could slow down the decline of cell proliferation activity caused by H 2 O 2 -induced acute OS, while it alone had no significant effect on cell proliferation (Figure 6(c)). Moreover, the overexpression of PAGE4 alone exerted no effect on SOD1, CAT, and ROS accumulation, but it ameliorated H 2 O 2 -induced acute OS (Figures 6(d)-6(h), Figure S2).

The Upregulation of PAGE4 Attenuated OS-Induced Cell
Apoptosis, GO/G1 Phase Arrest, and Activation of JNK Pathway, but Enhanced OS-Induced Activation of the ERK Pathway. Similar to PAGE4 knockdown, PAGE4 overexpression alone exhibited no effect on cell apoptosis and cell cycle but it reversed the H 2 O 2 -induced increase in apoptosis, GO/G1 phase arrest, and the corresponding G2 phase shortening (Figures 7(a)-7(d)), as well as related proteins (BAX, Bcl-2, Cyclin D1, and CDK6) (Figure 7(e)). Finally, Western blot revealed overexpression of PAGE4 could potentiate the increase of p-ERK1/2 and offset the increase of p-JNK1/2 triggered by H 2 O 2 (Figure 8).

Discussion
The current study determined that PAGE4 was upregulated in hyperplastic prostate and it was mainly localized in the stromal compartment. We further demonstrated that the augmented PAGE4 could promote cell survival by activating the phosphorylation of ERK1/2 and decrease cell apoptosis by inhibiting the phosphorylation of JNK1/2 under the  Although PAGE4 is a prostate-specific protein, it is highly dynamic. It is richly expressed in the prostate of the fetus at 21 weeks of gestational age, but almost undetectable after 36 weeks [15], and can be reupregulated in some benign and malignant prostate diseases. The current study demonstrated the richly expressed PAGE4 in BPH tissue both at mRNA and protein levels, while it was barely detectable in normal adult prostate. Of interest, it was predominantly localized in the stroma. Previous studies have shown that PAGE4 was located in mitochondria and that the expression level of PAGE4 was related to OS. In the aged prostate, cells are often subjected to severe OS due to obesity [23], smoking [24,25], and aging [26]. It has been reported that the level of OS markers was significantly increased in the plasma [27,28] and urine [29] specimens of patients with BPH. Thus, OS could contribute to BPH via modulating the PAGE4 pathway.
In order to simulate the environment of oxidative stress, we created an oxidative stress cell model induced by 400 μM H 2 O 2 , which was near its IC50 (IC50 WPMY-1 : 431.9 μM,  [30]. When ROS production is not coor-dinated with the biological system to repair oxidative damage, the direct effect of excessive ROS can inhibit cell proliferation and promote cell apoptosis via oxidative killing [31]. However, knockdown or overexpression of PAGE4 alone had no effect on cell growth and cell cycle as well as intracellular OS markers. Interestingly, H 2 O 2 combined with PAGE4 silencing potentiated intracellular OS, cell apoptosis, along with arrest of cell cycle, and inhibited cell proliferation. Parallelly, H 2 O 2 combined with PAGE4 overexpression ameliorate the aforementioned process. Therefore, our study 14 Oxidative Medicine and Cellular Longevity indicates that PAGE4 was upregulated in the presence of OS which lead to reduction of intracellular OS levels and promotion of cell survival. When the level of oxidative stress in the environment reached a certain threshold, PAGE4 was activated as a stress response protein to reduce OSinduced damage [17]. We further explored the possible mechanisms of OS-PAGE4 on the development of BPH. It is well known that MAPK signaling pathways regulate cell physiology via various pathways, including cell proliferation, differentiation, mitosis, cell survival, and apoptosis [32,33]. Indeed, our previous studies have shown that the MAPK signaling pathway is related to the development of BPH [13]. It is also been demonstrated that the activation of JNK1/2, a downstream gene of the MAPK pathway [34,35], can increase apoptosis. Additionally, another MAPK family member ERK1/2 participates in cell cycle regulation [36,37]. In the present study, the MAPK/JNK pathway and the MAPK/ERK pathway were activated after H 2 O 2 treatment. Meanwhile, H 2 O 2 combined with silencing PAGE4 enhanced the activation of JNK1/2 and weakened the activation of ERK1/2. On the contrary, H 2 O 2 combined with overexpression of PAGE4 weakened the activation of JNK1/2 and enhanced the activation of ERK1/2. It is speculated that oxidative stress induces cell death partly through activating the JNK pathway which has been known to be able to trigger an apoptotic cascade [38,39]. In contrast, the ERK pathway that promotes cell survival is also activated under OS, acting in conjunction with the JNK pathway to balance cell death [40]. In this con-text, PAGE4 protect cells from oxidative stress potentially by reducing cell death while enhancing cell survival. Lv et al. [17] also demonstrated the large number of colocalization sites of activated ERK1/2 and PAGE4 in human prostate tissues [41]. Considering that PAGE4 does not exist as a kinase, its activation of the MAPK signaling pathway would be indirect. OS was not only a direct activator of the JNK pathway [42,43] but also an indirect activator of the ERK pathway through vascular endothelial growth factor [44]. In addition, there was abundant signal cross-talk between the JNK and ERK pathways, and it was likely that the cross-activation of ERK and JNK contributed to the induction of cell division or eventual differentiation [45]. Therefore, long-term chronic oxidative stress, as an intermediary between PAGE4 and the MAPK signaling pathway, produced indirect and direct activation effects on ERK and JNK pathways, respectively. In addition, SOD1 and CAT, as antidotes of ROS, had antagonistic effects against OS. Actually, ROS levels were in dynamic balance with antioxidants such as SOD1 and CAT. When ROS levels were abnormally elevated, this balance was disrupted, resulting in decreased SOD1 and CAT expression, increased ROS accumulation, and activation of ERK and JNK pathways. On the other hand, the anti-OS function of PAGE4 needed to be activated in the presence of OS, upregulating SOD1 and CAT, thereby reducing ROS levels, activating p-ERK1/ 2, and inhibiting p-JNK1/2 in the regulation of the ERK-JNK interactive network. Finally, PAGE4 inhibited cell apoptosis and promoted cell proliferation. As one of the      Histopathologically, BPH is characterized by nodular hyperplasia of epithelial and stromal components, and the stromal-epithelial interaction mediated by paracrine signals between the epithelium and stromal is an important factor. Prostatic stromal cells can secrete growth factors (such as fibroblast growth factor and insulin-like growth factor I and II) and other cytokines (IL-2, IL-4, and IFN-γ) to regulate the proliferation and differentiation of epithelial cells and stroma itself [46,47]. As an abnormally expressed gene in the stroma of hyperplastic prostate, PAGE4 promotes the survival of stromal cells under OS, and the latter may affect  the epithelial cell proliferation of BPH through stromalepithelial interaction. Its specific mechanism needs to be further studied.
The limitation of the current study is using the acute OS cell model used to mimic the chronic OS occurring in the hyperplastic prostate. At present, there is no suitable chronic OS cell model for prostate cell signaling.

Conclusion
Our study demonstrates that PAGE4 enhances the survival of prostate stromal cells under oxidative stress. PAGE4 was activated via the stimulation of ROS production, promoting cell survival and reducing cell apoptosis by increasing the phosphorylation of ERK1/2 and decreasing the phosphorylation of JNK1/2, finally leading to the development and progression of BPH. Therefore, the high expression of PAGE4 in the pathologic prostate can be a potential novel therapeutic target for BPH.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon reasonable request.

Disclosure
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.