Fifty-nine BPH specimens of transition zone were collected from patients undergoing transurethral resection of the prostate at the Beijing Chaoyang Hospital, Capital Medical University. Patient characteristics are shown in Table 1. All human specimens were acquired under the approval of the Institutional Review Board of Beijing Chaoyang Hospital, Capital Medical University (2017-KE-6, Beijing, China). The research was executed according to the World Medical Association Declaration of Helsinki, and each patient signed written informed consent. The mean age of the BPH patients was 70 years, ranging from 56 -91 years. Before being evaluated for the expression of SRD5A2 protein, these specimens were paraffin-embedded and proven to be BPH and non-cancerous through routine histological analysis by pathologists.
2.2 Immunohistochemical analysis of SRD5A2
Immunohistochemistry (IHC) was performed as previously described by Lin et al9. Briefly, specimen sections were incubated with SRD5A2 primary antibody (Novus Biological Inc., Centennial, CO, USA，NBP1-46510) following the manufacturer’s recommendations at a concentration of 1/1500. Negative controls were used throughout the immunostaining protocol. Three representative areas from each sample were randomly selected under 40× magnification to assess immunoreactivity by two genitourinary pathologists. A hundred cells selected randomly from the epithelium were manually counted from each representative section. Each cell was scored on a 0–3 scale according to the intensity of the staining. Then, a visual score was generated for each sample, ranging from 0 to 300. A score of 0–100 was defined as weak expression, and a score of 101–300 indicated strong expression.
2.3 Cells and culture condition
The immortalized human prostatic epithelial cell line BPH-1 and HEK293T were obtained from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Normal human prostatic epithelial cell line RWPE-1 was acquired from Shanghai Zhong Qiao Xin Zhou Biotechnology Co.,Ltd. BPH-1 were cultured in RPMI 1640 medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Melbourne, Australia) and 1% penicillin -streptomycin (HyClone, Logan, UT, USA). 293T were cultured in Dulbecco's Modified Eagle Medium (Gibco, Rockville, MD, USA) supplemented with 10% FBS and 1% penicililin -streptomycin. RWPE-1 was cultured in Keratinocyte Medium with a keratinocyte growth supplement. Cells were incubated at 37℃ with 5% CO2. All in vitro experiments were repeated three times.
2.4 RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol reagent (Invitrogen Inc.,Carlsbad, CA, USA). The quality and quantity of extracted RNA were assessed using NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcriptase was used to produce the first-strand complementary DNA (TIANGEN, Beijing, China) according to the manufacturer's instructions. miRNA Real-Time PCR Assay kit was used to detect miRNA expression level (TransGen, Beijing, China). mRNA expression was normalized to GAPDH expression, whereas miRNA was normalized to U6. The relative RNA expression was calculated as the inverse log of the delta/delta CT. Specific primers are shown in Table 2.
2.5 Cell transfection
miR-1199-5p mimics, miR-100-5p inhibitor, and miRNA negative control (miR-NC) were synthesized by Suzhou GenePharma Co., Ltd. (Suzhou, China) and transfected into cells by siRNAmate Suzhou GenePharma Co., Ltd. (Suzhou, China) according to the manufacturer’s instructions. The RNA was extracted for qRT-PCR after transfection for 24h, and the protein was extracted for western blotting after transfection for 48h.
2.6 Western blotting
Cells were lysed for extracting total protein with RIPA buffer containing a mixture of protease and phosphatase inhibitors. The protein concentration was detected by the PierceTM BCA protein assay reagent (Thermo, Rockford, USA). 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel was used to separate the protein lysates. Then, they were transferred to poly (vinylidene fluoride) (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked and incubated with primary antibody of SRD5A2(1:1000) or GAPDH (1:2000) at 4 ℃ overnight. Subsequently, the PVDF membranes were washed three times with TBST and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000; Abcam, Cambridge, UK) at room temperature for 1 h. Then, the membranes were washed again three times for 5 mins each with TBST. Enhanced chemiluminescence (ECL) western blotting substrate was used for visualization and detection.
2.7 Dual-luciferase reporter assay
For the dual-luciferase reporter assay, 293T cells were seeded in 24-well plates (50 000 per well) and co-transfected with a PGL-3 control plasmid, wild-type or mutant SRD5A2 3ʹUTR vector, and miR-1199-5p mimics or control (Suzhou GenePharma Co., Ltd, Suzhou, China). After 24 h, the cells were harvested, and Firefly/Renilla luciferase activities were analyzed using the dual-luciferase reporter assay kit according to the manufacturer's protocol (Promega, Madison, WI, USA).
2.8 Flow cytometry analysis assay
Annexin V-FITC and PI Detection Kit (BD Biosciences, NJ, USA) was used to determine cell apoptosis. 2×105 RWPE-1 or BPH-1 cells treated with finasteride/DMSO and mimics/inhibitor were harvested, resuspended in 100μl flow cytometry binding buffer, and then stained with 5μl Annexin V/FITC and 5μl PI following manufacturer’s instructions. Determining BPH-1 and RWPE-1 cells apoptosis using flow cytometry (BD FACSCantoTM II, NJ, USA).
2.9 Statistical analysis
All statistical analyses were performed using SPSS 23 software (SPSS, Chicago). Comparisons between datasets from qRT-PCR, western blotting and fluorescence analysis were carried out using a t-test. All tests were two tailed, and P < 0.05 was considered statistically significant.