Prostate tissue samples were collected from 5 patients with BPH and 5 healthy volunteers (aged 42±10 years) at The Second Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China) between December 2016 and January 2017. The participants included in the current study had not received any medical treatment prior to tissue collection. A total of 10 prostate tissue samples were immediately snap-frozen in liquid nitrogen following radical prostatectomy. Extracted RNA was used for microarray analyses and RT-qPCR. An additional 25 BPH samples (aged 43±8 years) were included in the RT-qPCR assay between January 2017 and April 2017 at The Second Affiliated Hospital, Zhejiang University School of Medicine. Clinicopathological information for each patient was obtained, including age and levels of preoperative prostate-specific antigen. The present study was conducted with the approval of The Ethics Review Board at Zhejiang University School of Medicine (Hangzhou, China) and all participants provided written informed consent.

WPMY-1 cells obtained from the cell bank at the Chinese Academy of Sciences (Shanghai, China) were cultured in DMEM medium (11965–092; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 100 U/ml penicillin/streptomycin and 10% fetal bovine serum (15140–122 and 10099–141 both from Gibco; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂, as previously described (16).

RNA was extracted with the miRNeasy Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. A NanoDrop ND-1000 spectrometer (Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA) was used to detect the RNA yield and 260/280 nm ratio. A 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) was used to quantify the RNA integrity number.

Differential expression analysis of miRNAs and mRNA between the pooled samples was performed using Affymetrix MiRNA Microarray technology version 4.0 (Affymetrix; Thermo Fisher Scientific, Inc.) (17) and GeneChip Human Transcriptome Array 2.0 (Thermo Fisher Scientific, Inc.) (18,19).

A TaqMan miRNA assay (Thermo Fisher Scientific, Inc.) was used to detect mature miRNAs using a Bio-Rad IQ5 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol, as previously described (6). The reactions were performed using the following parameters: 95°C for 2 min followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. RNU6-1 small nuclear RNA was used as an endogenous control for data normalization. Relative expression was calculated using the comparative threshold cycle method (20). Experiments were performed in triplicate. RT-qPCR analyses for the mRNA expression of target genes were performed using PrimeScript RT-PCR kits (Takara Biotechnology Co., Ltd., Dalian, China). The reactions were performed using the following parameters: 95°C for 2 min followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec, 72°C for 20 sec, and then 72°C for 10 min. β-actin was used as an internal control (20). The primer sequences used are presented in Table I.

In order to further understand the functions of miRNA and their targets, GO enrichment and KEGG pathway analysis for all annotated miRNAs and their targets were performed. Blast2GO was employed to store information from the GO (http://www.geneontology.org/) and KEGG (http://www.kegg.jp) pathway databases. All the sequences were identified by BLASTx searches against the GO protein database. A combined query was used in order to complete the GO annotation and pathway analysis against the GO and KEGG databases (21).

Mimic and control miR-96-5p were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China) and transfections were performed with DharmaFECT 1 transfection reagent (Thermo Fisher Scientific, Inc.). WPMY-1 cells were transfected with 100 nM mimic or miRNA control for 48 h, and then mRNA expression was subsequently assessed by RT-qPCR.

Data are expressed as the mean ± standard error of the mean. Statistical analysis was performed using the Student's t-test for comparison of two groups in microarray analysis, and analysis of variance for comparisons of expression of selected aberrant miRNAs in RT-qPCR assay, using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the miRNA expression profiles in the BPH and control group, array-based miRNA profiling of the prostate tissues from patients with BPH was performed. Of the 1,872 human miRNAs assayed, 23 miRNAs including miR-4428, miR-106a-5p, miR-96-5p and miR-1271-5p had higher expression levels in the prostate of patients with BPH when compared with healthy controls. The expression levels of 15 miRNAs including miR-16-5p, miR-19b-5p, miR-940, and miR-25 were reduced in the prostate of patients with BPH compared with healthy controls (Fig. 1). Of these 26 miRNAs, miR-96-5p and miR-1271-5p were significantly overexpressed, whereas miR-30a was significantly downregulated in the prostates of men with BPH.

To confirm the microarray findings, the relative expression of each dysregulated miRNA was examined with miRNA RT-qPCR in all 30 BPH samples and the 5 healthy controls. The expression levels of hsa-miR-96-5p, hsa-miR-1271-5p, hsa-miR-21-3p, hsa-miR-96-5p, hsa-miR-181a-5p, hsa-miR-143-3p, hsa-miR-4428 and hsa-miR-106a-5p were significantly upregulated in the BPH group compared with healthy controls. The expression levels of hsa-miR-16-5p, hsa-miR-19b-5p, hsa-miR-940, hsa-miR-25, hsa-miR-486-3p, hsa-miR-30a-3p, hsa-miR-let-7c and hsa-miR-191 were significantly downregulated compared with the control (Fig. 2). These findings were in concordance with the microarray assay results. Notably, hsa-miR-96-5p and hsa-miR-1271-5p have the same seed sequence and were classified in the same cluster.

Gene ontology and KEGG pathway analysis of the signaling pathways regulated by miRNAs (as identified by RT-qPCR) revealed 47 upregulated signaling pathways and 28 downregulated signaling pathways in the BPH group compared to healthy controls. These regulated signaling pathways were involved in numerous cellular processes, including metabolic (pyrimidine, glutathione, phenylalanine, gluconeogenesis, and pyruvate), oncogenic (prostate cancer, colorectal cancer, and bladder cancer) and adhesive and signal transduction [estrogen, phosphoinositide 3-kinase (PI3K-AKT), Wnt, transforming growth factor β (TGF-β) and mitogen-activated protein kinase (MAPK)] signaling pathways. In addition, according to the enrichments of the significantly downregulated signal genes, there were 28 downregulated signaling pathways (Fig. 3).

The miRNA-gene network of overlapping target genes identified the involvement of hsa-miR-96-5p (Fig. 4A). The major target genes of hsa-miR-96-5p were identified by TargetScan version 7.1 (www.targetscan.org), include mechanistic target of rapamycin (MTOR), RPTOR independent companion of MTOR complex 2 (RICTOR), syntaxin 10 (STX10), growth receptor bound protein 2 (GRB2), autophagy related 9A, zinc finger E-box binding homeobox 1 (ZEB1), caspase 2 (CASP2) and protein kinase c ε (PRKCE). In order to determine whether these genes were miR-96 target genes, WPMY-1 cells were transfected with miR-96-5p mimics or miR-96-5p control. As presented in Fig. 4B, miR-96 had an inhibitory effect on the mRNA expression level of these target genes, excluding GRB2. This verified that miR-96 may downregulate these target genes.