This study was approved by the Ethical Committee of Ningbo No. 2 Hospital. Written informed consent was provided by all patients prior to enrollment in this study. A total of 27 pairs of PCa tissues and adjacent noncancerous tissues (NCTs) were obtained from patients who underwent surgery at Ningbo No. 2 Hospital from June 2014 to February 2016. None patients had been treated with any treatment including chemotherapy, radiotherapy and androgen-deprivation treatment before surgery. Following surgical resection, all tissues were immediately frozen in liquid nitrogen and stored at −80°C until further use.

Human PCa cells (DU145, PC3, LNCaP and 22RV1) and normal prostatic epithelial cell line (RWPE-1) were acquired from Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China). All PCa cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin (all from Gibco, Grand Island, NY, USA). RWPE-1 cells were grown in keratinocyte-serum-free medium containing 50 mg/ml bovine pituitary extract and 5 ng/ml recombinant human epidermal growth factor (all from Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All cell lines were maintained at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.

miR-136 mimics and negative control miRNA mimics (miR-NC) were purchased from GenePharma Co., Ltd. (Shanghai, China). MAP2K4 overexpression plasmid (pCMV-MAP2K4) and blank plasmid pCMV were chemically synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). For transfection, cells were plated in 6-well plates 24 h prior to transfection. Cell transfection was carried out using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions.

Total RNA was isolated from tissues or cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's instructions. A NanoDrop 2000 (Thermo Fisher Scientific, Inc., Wilmington, DE, USA) was used to determine the RNA purity and concentration. To quantify miR-136 expression, the complementary DNA (cDNA) was synthesized using the TaqMan MicroRNA Reverse Transcription kit and the quantitative PCR was performed using the TaqMan MicroRNA PCR kit (both from Applied Biosystems, Foster City, CA, USA). U6 snRNA used as an control for miR-136 expression. For detection of MAP2K4 mRNA, a PrimeScript RT Reagent kit (Takara Biotechnology, Co., Ltd., Dalian, China) was utilized to synthesize the cDNA. The PCR amplification for the quantification of MAP2K4 mRNA was carried out using the SYBR Premix Ex Taq^™ kit (Takara Biotechnology, Co., Ltd.). The relative expression of MAP2K4 mRNA was illustrated as fold difference relative to GAPDH. Relative expression was analyzed using the 2^−ΔΔCq method (21).

PCa cell proliferation was detected by the CCK-8 assay in accordance with the manufacturer's instructions. Transfected cells were collected 24 h post-transfection. For the CCK-8 assay, cells were seeded in 96-well plates at a density of 3×10³ cells/well. The CCK-8 assay was performed at 0, 24, 48, 72 and 96 h after incubation. In brief, 10 µl of the CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan) was added, and the plate was incubated at 37°C with 5% CO₂ for 2 h. Absorbance at 450 nm wavelength was recorded on a multilabel plate reader (BioTek Instruments, Inc., Winooski, VT, USA). Each assay was performed in triplicate and repeated three times.

The invasion ability of PCa cells was examined using Transwell chambers (EMD Millipore, Billerica, MA, USA) coated with Matrigel (BD Biosciences, San Jose, CA, USA). The transfected cells were harvested 48 h post-transfection by using 0.2% trypsin/EDTA solution (Gibco) and washed with fetal bovine serum (FBS)-free RPMI-1640 medium. A total of 5×10⁴ transfected cells in FBS-free RPMI-1640 medium were added to the upper chamber. RPMI-1640 medium supplemented with 10% FBS was then added to the lower chamber to serve as chemoattractant. After incubation at 37°C with 5% CO₂ for 24 h, the cells attached to the upper surface of the chamber were removed by a cotton swab. The invasive cells were fixed with 100% methanol and stained with 0.5% crystal violet solution (Beyotime Institute of Biotechnology, Haimen, China). The invasive cells were then photographed and counted in five randomly selected fields under an inverted microscope (×200; Olympus, Tokyo, Japan).

Potential target genes for miR-136 were predicted using (http://www.targetscan.org/index.html) and miRanda (http://www.microrna.org/microrna/).

For luciferase reporter assay, the luciferase plasmids, pmirGLO-MAP2K4-3′-UTR wild-type (Wt1 and 2) and pmirGLO-MAP2K4-3′-UTR mutant (Mut1 and 2), were chemically synthesized and confirmed by GenePharma Co., Ltd. Cells were seeded into 24-well plates at a density of 1.5×10⁵ cells each well and cultured at 37°C with 5% CO2 for 24 h. miR-136 mimics or miR-NC was transfected into cells, together with pmirGLO-MAP2K4-3′-UTR Wt (1 and 2) or pmirGLO-MAP2K4-3′-UTR Mut (1 and 2), using Lipofectamine 2000 according to the manufacturer's instructions. At 24 h post-transfection, luciferase activities were measured by using a Dual-Luciferase Reporter Assay system (Promega, Madison, WI, USA), according to the manufacturer's instructions. Renilla luciferase activity was used as an internal control.

Total protein was extracted from tissues or cells using RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China). After centrifugation at 12,000 × g for 15 min at 4°C, a BCA Protein Assay kit (Beyotime Institute of Biotechnology) was adopted to detect the protein concentration. Equal amounts of protein was loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (EMD Millipore). After blocking with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) at room temperature for 2 h, the membranes were incubated at 4°C overnight with primary antibodies: Mouse anti-human MAP2K4 monoclonal antibody (sc-136314, 1:1,000 dilution) or mouse anti-human GAPDH monoclonal antibody (sc-47724, 1:1,000 dilution) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Afterwards, the membranes were washed with TBST for three times and probed with goat-anti mouse horseradish peroxidase-conjugated secondary antibody (sc-2005, 1:5,000 dilution; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Finally, the proteins were visualized using ECL Immunoblot Detection system (Pierce, Rockford, IL, USA). Densitometry of protein bands was quantified with Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH served as a loading control.

Data are presented as the median ± standard deviation, and analyzed using SPSS software (version 20.0; SPSS Inc., Chicago, IL, USA). Differences between groups were analyzed using student's t-test or one-way ANOVA with a Student-Newman-Keuls post hoc test. The association between miR-136 and MAP2K4 expression levels was determined using Spearman's correlation analysis. P<0.05 were considered to indicate a statistically significant difference.

To investigate the expression pattern of miR-136 in PCa, we firstly measured the expression level of miR-136 in 27 pairs of PCa tissues and NCTs. Results showed that miR-136 expression was downregulated in PCa tissues than in NCTs (Fig. 1A; P<0.05). In addition, miR-136 expression was detected in four PCa cell lines (DU145, PC3, LNCaP and 22RV1) and a normal prostatic epithelial cell line (RWPE-1). The expression level of miR-136 was lower in all examined PCa cell lines than in RWPE-1 (Fig. 1B; P<0.05). These results suggested that the decreased expression of miR-136 was likely associated with PCa progression.

To evaluate the biological roles of miR-136 in PCa, we transfected DU145 and PC3 cells, which were expressed relatively lower miR-136 levels, with miR-136 mimics to increase miR-136 expression levels. After transfection, RT-qPCR confirmed that transfection with miR-136 mimics significantly increases the miR-136 levels relative to those of the cells transfected with miR-NC (Fig. 2A; P<0.05). Subsequently, CCK-8 assay was performed to investigate the effect of miR-136 overexpression on PCa cell proliferation. Results revealed that upregulating miR-136 significantly decreases the growth rate relative to that in the miR-NC-transfected DU145 and PC3 cells at 48 and 72 h after plating (Fig. 2B; P<0.05). We also examined the effect of miR-136 on cell invasion ability by using a Matrigel invasion assay. The ectopic expression of miR-136 decreased the invasion capacities of both DU145 and PC3 cells relative to those in the miR-NC group (Fig. 2C; P<0.05). These results suggest that miR-136 is involved in regulating PCa progression and may serve as a tumour suppressor.

To study the mechanism underlying the tumour-suppressive roles of miR-136 in PCa, we conducted bioinformatics analysis to predict the potential targets of the miRNA. MAP2K4, which is highly expressed in PCa and contributes to PCa occurrence and development (22–24), has been predicted as a major target of miR-136 and selected for further confirmation. The 3′-UTR of MAP2K4 contains two predicted binding sites for miR-136 (Fig. 3A). To determine whether miR-136 can directly interact with the 3′-UTR of MAP2K4, we conducted a luciferase reporter assay. Results showed that miR-136 overexpression significantly repressed the activity of the MAP2K4-3′-UTR luciferase plasmid containing a wild-type gene (1 and 2; Fig. 3B and C; P<0.05). By contrast, no change in luciferase activity was observed when the miR-136 binding site was mutated (1 and 2). This observation indicates that miR-136 directly interacted with the two target regions in the 3′-UTR of MAP2K4. RT-qPCR and western blotting were performed to illustrate that miR-136 can regulate the endogenous expression of MAP2K4. Our data showed that the enforced expression of miR-136 reduced the MAP2K4 expression in DU145 and PC3 cells at the mRNA (Fig. 3D; P<0.05) and protein (Fig. 3E; P<0.05) levels. These findings suggest that MAP2K4 is a direct target of miR-136 in PCa.

To further explore the relationship between miR-136 and MAP2K4 in PCa, we detected MAP2K4 expression levels in PCa tissues and NCTs. RT-qPCR results indicated that MAP2K4 mRNA expression was significantly increased in the PCa tissues than in the NCTs (Fig. 4A; P<0.05). In addition, the protein expression level of MAP2K4 was examined in four randomly selected paired PCa tissues and NCTs. The data of western blotting revealed that protein level of MAP2K4 was higher in the PCa tissues than in the NCTs (Fig. 4B and C; P<0.05). A negative correlation was observed between miR-136 and MAP2K4 mRNA expression in PCa tissues (Fig. 4D; r=−0.6113, P=0.0007). This negative correlation suggests that the upregulation of MAP2K4 in PCa may partially be due to the downregulation of miR-136.

To investigate whether miR-136 exerts its tumour suppressive roles in PCa by regulating MAP2K4, we carried out a rescue experiment involving DU145 and PC3 cells cotransfected with miR-136 mimics and pCMV or pCMV-MAP2K4. After transfection, Western blotting analysis revealed that the reduced expression of MAP2K4 induced by miR-136 mimics can be significantly recovered by cotransfection with pCMV-MAP2K4 in DU145 and PC3 cells (Fig. 5A; P<0.05). Subsequently, functional assays showed that restored MAP2K4 expression can reverse the inhibitory effects of miR-136 overexpression on the proliferation (Fig. 5B; P<0.05) and invasion (Fig. 5C; P<0.05) of DU145 and PC3 cells. These results clearly demonstrated that miR-136 can regulate the malignant biological behaviour of PCa cells by inhibiting MAP2K4.