Prostate cancer samples and the adjacent normal tissues were collected from 58 patients who underwent tumor-resection surgery in Peking Union Medical College Hospital from September 1, 2014 to January 1, 2016. Patients signed an informed consent form. The tissues were separately frozen in liquid nitrogen for further study. Protocols were approved by the institutional ethics committee of Peking Union Medical College Hospital and carried out in accordance with the guidelines for ethical management. PCa cell lines, PC3 and DU145, and prostate epithelial cells were purchased from American Type Culture Collection (ATCC, USA). PC3 was incubated in F-12 medium, and DU145 was grown in DMEM medium (Life Technologies, Inc., USA), and all the mediums were supplemented with penicillin/streptomycin (100 U/ml and 100 mg/ml, respectively) and 10% fetal bovine serum (Life Technologies Inc.). The human prostate epithelial cells (hPrEC) were cultured in prostate epithelium basal media. All the cells were incubated at 37°C in a humidified atmosphere of 5% CO₂.

Total RNA was extracted from the tissues or cells via TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and quantified with UV absorbancy at 260 and 280 nm (A260/280). Subsequently the RNA was reverse-transcribed into cDNA by using reverse transcription system (Thermo Scientific, CA, USA). The expression level of miR-212 was detected by the Applied Biosystems 7500 Real-time PCR system (ABI) using the TaqMan MicroRNA assay kits (Applied Biosystems, CA, USA). U6 was used as the control normalizer. The gene expression of EN-2 was also analyzed by SYBR Green and normalized with GAPDH. The specificity of primer sequences was analyzed by dissociation curve, 2^−ΔΔCt (cycle threshold) was used to calculate the relative gene expression levels.

Protein from tissues or cells was collected by using RIPA buffer which contain a protease inhibitor cocktail and phosphatase inhibitors (Sigma, St. Louis, MO, USA), according to the manufacturer's instructions and quantified via bicinchoninic acid (BCA) kit (Thermo Scientific). Protein samples (30 µg) were separated by SDS-PAGE and then transferred onto the polyvinylidene difluoride (PVDF, Millipore, Bedford, MA, USA) membranes. Western blotting was performed using anti-β-actin, anti-EN-2, anti-Bcl-2 and anti-Bax (CST, Denver, CO, USA). The protein levels were detected with an ECL kit (Thermo Scientific) according to the manufacturer's instructions.

CCK-8 assay was performed to evaluate cell proliferation according to the manufacturer's instructions. For experiments the cells were seeded at a density of 5×10³ cells per well in 96-well plates and cultured for various periods of time (0, 24, 48 and 72 h). The absorbance at 450 nm was measured using an electroluminescence immunosorbent assay reader (Thermo Fisher Scientific, Waltham, MA, USA).

Cells were collected and washed twice with cold phosphate-buffered saline solution (PBS) to remove floating cells then labeled with Annexin V-FITC (BD Biosciences, San Jose, CA, USA). Apoptosis were evaluated with a flow cytometry analyzer. Data were analyzed by Flowjo 7.6 software.

Cells were fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, and incubated with the TUNEL detection kit from Roche (Roche, Shanghai, China) at 37°C for 1 h. The samples were mounted in mounting media containing DAPI. Fluorescent images were captured using a fluorescence microscope. The total number of DAPI positive cells and total number of TUNEL positive cells were counted.

Cell invasion assays were performed by using 24-well Transwell plates (BD Biocoat, USA). Cells transfected with the miR-212 mimics and mimic control for 24 h. Then the cells were plated onto the 24-well upper chamber with a membrane that was pre-treated with Matrigel (100 µg per well; BD Biosciences). In the lower portion of the chamber, fresh medium containing 10% FBS was added. After the cells were incubated for 24 h at 37°C, the cells in the upper chamber were removed. Invaded cells were fixed with 4% formaldehyde, stained with 0.5% crystal violet, and counted under a microscope.

Cells were seeded into 6-well plates. Wounds were created in the confluent cell monolayer using a 200-µl sterile pipette tip, and the other free-floating cells and debris were removed by washing with PBS three times. Medium was then added, and the culture plates were incubated at 37°C for another 24 or 48 h. Wound healing within the scraped wound line was measured at a 48-h time-point by using microscope (Nikon, Tokyo, Japan).

TargetScan software (http://www.targetscan.org/) was used to identify the potential target gene of miR-212. The putative miR-212 binding sites or mutant at the 3′-UTR of EN-2 were cloned into a vector and EN-2 3′-UTR WT or EN-2 3′-UTR Mut, was generated, respectively. EN-2 3′-UTR WT or EN-2 3′-UTR Mut was transfected into PCa cells which stably overexpressed miR-212 and transfected with negative control. Dual-Luciferase Reporter assay system (Promega, Madison, WI, USA) was used to detect the luciferase activity 36 h after transfection.

The LV-miR-212 mimics and mimics control were purchased from Gene-Chem (Shanghai, China). Cells were seed into 24-well plates at a density of 30% and cultured overnight before transfection. Cells were transfected with miR-212 mimics or mimics control by using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocols.

In this study all the animal procedures and experiments were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. The 6–8-week-old nude male mice [BALB/c A-nu (nu/nu)] were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed in the Animal Resource Facility at Laboratory Animal Centre. Cells stably expressing either miR-212 overexpression (LV-miR-212) or miRNA control were injected subcutaneously into the mice. The volume of the tumor was recorded for 7 weeks (once each week).

All quantitative data presented are the mean ± SD from at least three independent experiments. The relationship between the expression level of miR-212 and clinicopathological parameters was examined using Fisher's exact test or χ² test. One-way ANOVA test or Student's t-test was used to analyze the other date. The significant statistical differences level was set at P<0.05. SPSS 18.0 and Graph Pad Prism 6.0 software were used for date analysis.

In order to confirm the molecular mechanisms underlying PCa, we analyzed the expression level of miR-212 and EN-2 in a series of clinical PCa tissues. Results showed that miR-212 was markedly decreased in cancer samples when compared with the adjacent normal tissues (Fig. 1A). In contrary, the level of EN-2 mRNA was over-expressed (Fig. 1B). Subsequently, we measured the miR-212 and EN-2 level in PCa cell lines and the human prostate epithelial cells (hPrEC) by RT-qPCR. As shown in Fig. 1C, the expression level of miR-212 in PCa cells was significantly lower than that in hPrEC cells. While the expression level of EN-2 had the opposite trend (Fig. 1D). The relationship between miR-212 expression level and clinicopathological features in our PCa cases is presented in Table I. The decreased miR-212 was significantly associated with high Gleason scores (P<0.05). As shown in Table I, the decreased levels of miR-212 was present in 3 of 11 (27.3%) cases with low Gleason score (<7), 8 of 17 (47.1%) cases with Gleason score 7, but 21 of 30 (70.0%) cases with high Gleason score (>7). The expression level of miR-212 was obviously decreased in PCa patients with distant metastasis (P<0.05). There were no statistically significant difference between miR-212 level and the age of the patients, PSA levels or tumor stage.

Next, in order to verify the role of miR-212 in control PCa cell survival and progress, miR-212 mimics and miR-NC were transfected into DU145 cells, then cells were collected for analysis. The level of miR-212 was detected by qRT-PCR. Results showed that the expression level of miR-212 in the mimic group was markedly increased when compared with the control (Fig. 2). CCK-8 assay demonstrated that miR-212 upregulation significantly suppressed DU145 cells proliferation (Fig. 3A). Moreover, the TUNEL and flow cytometry assays demonstrated that the apoptosis of DU145 cells was increased when transfected with miR-212 mimics (Figs. 3B and C and 4A), whereas, the protein expression levels of Bcl-2 decreased and Bax markedly increased in DU145 cells after transfected with miR-212 mimics (Fig. 4B). Subsequently, we measured the effect of miR-212 on cell invasion, the Transwell assays indicated that miR-212 mimics transfected DU145 cells markedly suppressed cell invasion ability when compared to the mimic control cells (Fig. 3D and E). A similar tendency was discovered in the wound healing assays (Fig. 5). These outcomes clearly suggested that restoration of the expression of miR-212 inhibits PCa cell proliferation, invasion and migration.

In order to illustrate that EN-2 is a direct target of miR-212, EN-2 wild-type (WT) or mutant 3′-UTR (Mut) (Fig. 6A) were subcloned into a luciferase reporter vector and co-transfected with miR-212 mimics or negative control (NC) into PCa cells. Then, luciferase activity was detected after 48-h transfection. The results demonstrated that miR-212 markedly inhibited luciferase activity of the EN-2 WT 3′-UTR but had no influence on the mutant (Fig. 6B). To verify whether miR-212 can regulate the expression of EN-2, miR-212 mimics was transfected into DU145 cells for analysis. Results showed that the mRNA and protein level of EN-2 was markedly suppressed by miR-212 upregulation (Figs. 6C-E and 8).

In order to study the effect of EN-2 in PCa survival and invasion, miR-212 mimics and pcDNA-EN-2 were co-transfected into PCa cell lines, and after transfected for 48 h, cell survival and the ability of invasion were measured. Results showed that restoration of EN-2 reversed the anti-cell survival actions of miR-212 and the cell proliferation ability also recovered in comparison to miR-212 mimics (Figs. 3A-C and 4A). Moreover, the capability of invasion of PCa cells was significantly restored via upregulation of EN-2 when compared with miR-212 mimics (Fig. 3D and E), the migration ability of PCa cells was markedly increased by EN-2 restoration when compared with miR-212 mimics (Fig. 5).

In order to confirm the role of miR-212 against tumor growth in vivo, an animal model was made by nude mice subcutaneously injected with LV-miR-212. The tumor volume of subcutaneous xenograft tumors in nude mice apparently decreased with miR-212 upregulation when compared with the LV-control and the significant difference emerged from 4-week post-tumor transplantation (Fig. 7).