PC3, DU145 and LNCaP cells were obtained from Type Culture Collection of Chinese Academy of Sciences (Shanghai, China); PC3 is an aggressive prostate cancerous cell line whereas DU145 and LNCaP cells are non-aggressive prostate cancerous cell lines. The cells were maintained in F-12, modified Eagle's medium and RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc., USA) and penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The cells were maintained in an incubator containing 5% CO₂ at 37°C and 100% humidity. C4-2B cell lines were purchased from UroCor, Inc. (Shanghai, China) and maintained in RPMI-1640 supplemented with 10% FBS. miRNA-138 mimics (GCC GGA CUA AGU GUU GUG GUC GA; 50 nM) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) and β-catenin overexpression plasmid (100 nM) was obtained from Addgene Inc. (Cambridge, MA, USA), (Plasmid 17,198). Transient transfection was performed using the Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Assays were performed in BioCoat transwell chambers (BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA) with uncoated porous filters (pore size 8 µm) to evaluate cell migration, or Matrigel coated porous filters to exam cell invasion. C4-2B and PC3 cells (5×10⁵/ml) in serum-free medium were seeded into the upper chamber of each insert, and complete medium was added to the lower chamber. Following 12 h of incubation at 37°C, cells in the upper chambers were removed using cotton tips. Filters were fixed in 4% paraformaldehyde and stained at room temperature for 24 h with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) for counting. Independent experiments were repeated at least three times. Values for cell migration or invasion are expressed as the average number of cells per microscopic field (CX23 microscope; Olympus Corporation, Tokyo, Japan) over five fields for each filter.

Total RNA was extracted from t C4-2B and PC3 cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The relative expression levels of miR-138 were determined by RT-qPCR using mirVana™ qPCR microRNA Detection kit, according to the manufacturer's protocol. An Applied Biosystems^® 7500 thermal cycler, (Thermo Fisher Scientific, Inc.) was used and the reaction conditions were as follows: 95°C for 10 min, 40 cycles at 95°C for 15 sec and 60°C for 30 sec. For the PCR reaction, 1 µl cDNA solution, 10 µl PCR master mix, 2 µl of primers and 5 µl H₂O were mixed to obtain a final reaction volume of 20 µl. Specific primer sets for miR-138 and U6 were obtained from Genecopoeia (Applied Biosystems; Thermo Fisher Scientific, Inc.). The mRNA expression levels of LIMK1 were detected by RT-qPCR using the standard SYBR-Green RT-PCR kit (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) were as follows: miR-138; sense 5′-GCGAACTGTTTGCAGAGG-3′, antisense 5′-CAGTGCGTGTCGTGGAGT-3′; U6 forward, 5′-TGCGGGTGCTCGCTTCGGCAGC-3′ and reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′ and GAPDH forward, 5′-TGTGGGCATCAATGGATTTGG-3′ and reverse, 5′-ACACCATGTATTCCGGGTCAAT-3′. The relative expression levels miR-138 were quantified using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA), and the 2-ΔΔCq method (13).

Whole-cell extracts were obtained by lysis of cells in an appropriate volume of ice-cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). The BCA Protein Assay kit (Thermo Fisher Scientific, Inc.) was used to determine protein concentration. Protein samples (20 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to a polvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.) and blocked in phosphate-buffered saline/Tween-20 containing 5% non-fat milk for 1 h at room temperature. Subsequently, the PVDF membrane was incubated with mouse anti-E-cadherin antibody (ab1416; 1:200; Abcam, Cambridge, UK), mouse anti-vimentin antibody (ab8978; 1:200; Abcam), mouse anti-β-catenin antibody (ab22656; 1:200; Abcam), mouse anti-active-β-catenin antibody (05–665; 1:200; Sigma-Aldrich; Merck KGaA), anti-lamin B1 (ab8982; 1:200; Abcam) and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (ab8245; 1:50; Abcam) at 4°C overnight. The membranes were washed with TBS containing 0.1% Tween-20, and incubated with a corresponding horseradish peroxidase conjugated secondary antibody (ab6789; 1:3,000; Abcam) at 37°C for 1 h. Following extensive washing, proteins were visualized by enhanced chemiluminescence (ab5801; Abcam) and exposure to film (Fujifilm, Tokyo, Japan).

Cells were transiently transfected with 1 µg of a constitutively active vector encoding Renilla luciferase (Promega Corporation, Madison, WI, USA) and 10 µg of β-catenin-responsive firefly luciferase reporter plasmid TopFlash (EMD Millipore, Billerica, MA, USA) or the negative control FopFlash (EMD Millipore) using the Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells were harvested following 24 h in culture and firefly and Renilla luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega Corporation) according to the manufacturer's protocol. The firefly luciferase activity was normalized against the Renilla luciferase activity and fold increase in TOPFlash activity compared to FOPFlash is reported.

Data are presented as the mean ± standard deviation. Analysis was performed using SPSS software version 20.0 (IBM Corp, Armonk, NY, USA). All data were assessed using the Student's t-test or one-way analysis of variance followed by Newman Keuls post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To determine the role of miR-138 in prostate cancer, the present study firstly investigated miR-138 expression in six prostate cancer cell lines (LNCaP, C4/C5, C4-2, DU145, C4-2B and PC3) using RT-qPCR. It was demonstrated that miR-138 expression was associated with the aggressiveness, or, metastatic capabilities of tumor cell lines. In the prostate cancer cell lines C4-2B and PC3 with metastatic abilities, miR-138 expression levels were significantly decreased, compared with LNCaP, C4/C5, C4-2 and DU145 cell lines (Fig. 1). These results suggested that miR-138 expression may be associated with prostate cancer progression, and miR-138 may function as a tumor suppressor.

To further investigate the miR-138 function in prostate cancer, the present study transfected miR-138 mimics into C4-2B and PC3 cells. As presented in Fig. 2A, following transfection, miR-138 expression was significantly upregulated in the two cell lines. A Transwell assay was then performed to examine whether there was an invasive or migratory alteration following miR-138 overexpression. It was demonstrated that C4-2B and PC3 cells transfected with miR-138 mimics exhibited impaired invasion and migration (Fig. 2B and C), compared with transfected control miRNA. Epithelial marker E-cadherin expression was then investigated, and it was revealed that miR-138 overexpression induced E-cadherin expression in the two cell lines (Fig. 2D). Furthermore, expression of the mesenchymal marker vimentin was investigated, following miR-138 overexpression. Consistent with E-cadherin alteration, vimentin expression was downregulated (Fig. 2D). These results suggested that miR-138 may participate in tumor metastasis regulation.

The Wnt/β-catenin pathway is involved in tumor metastasis (14). To further investigate whether miR-138 affects the Wnt/β-catenin pathway in prostate cancer, the present study examined the Wnt/β-catenin pathway activation status. β-catenin dephosphorylation and translocation to the nucleus are the core events in Wnt/β-catenin pathway activation. β-catenin phosphorylation status was investigated. It was demonstrated that the active form of β-catenin (dephosphorylated) was downregulated in C4-2B and PC3 cells transfected with miR-138 mimics (Fig. 3A). To determine β-catenin localization, nuclear protein and cytoplasmic proteins from C4-2B and PC3 cell lines transfected with either miR-138 mimics or control mimics were extracted. Then, β-catenin expression was examined by immunoblot assay. In accordance with previous results, nuclear localization of β-catenin in C4-2B and PC3 cells transfected with miR-138 mimics was downregulated compared with controls (Fig. 3B). Furthermore, lymphoid enhancer factor/T-cell factor (LEF/TCF) transcription activity was examined via luciferase activity assay. Following miR-138 overexpression, C4-2B and PC3 cells indicated weakened LEF/TCF transcription activity (Fig. 3C). These results suggested that miR-138 suppresses the Wnt/β-catenin pathway in prostate cancer.

To further investigate whether the miR-138 anti-tumor effects were Wnt/β-catenin pathway dependent, β-catenin was overexpressed in C4-2B and PC3 cells, to activate the Wnt/β-catenin signaling pathway. It was then examined whether miR-138 overexpression induced an anti-tumor effect. As presented in Fig. 4A, β-catenin was overexpressed efficiently in C4-2B and PC3 cells. β-catenin overexpression restored C4-2B and PC3 cell invasion and migration capacity (Fig. 4B). Furthermore, E-cadherin and vimentin expression were examined following β-catenin overexpression. In accordance with previous results, the expression levels of the two proteins were restored (Fig. 4B). These results suggested that the miR-138 tumor suppressor role is dependent on Wnt/β-catenin pathway inhibition.