PCa cell lines LNCaP and C4-2 were obtained from the American Type Culture Collection (Manassas, VA, USA). BPH-1 cells were provided by Dr Jer-Tsong Hsieh (University of Texas Southwestern Medical Center, Dallas, TX, USA). These three cell lines were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) in a humidified chamber at 37°C in 5% CO₂.

Total RNA was extracted from harvested cells using TRIzol (Thermo Fisher Scientific, Inc.) and reverse transcribed to cDNA using the miScript II RT kit (Qiagen GmbH, Hilden, Germany), according to the manufacturers' protocols. The CFX96 PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with SYBR-Green PCR Master Mix (Takara Bio, Inc., Otsu, Japan) was used to detect the transcriptional expression of miR-218. The thermocycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, and then 60°C for 30 sec. U6 was used as an internal control, and relative gene expression was calculated using the 2^−ΔΔCq method (12). The primer sequences used were as follows: miR-218 forward, 5′-CGAGTGCATTTGTGCTTGATCTA-3′ and reverse, 5′-TAATGGTCGAACGCCTAACGTC-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-TGGTGTCGTGGAGTCG-3′.

LNCaP and C4-2 cells were seeded and cultured for 24 h and to 40-50% confluence. The lentiviral vector 3 (LV3)-miR-218, constructed by GenePharma Co., Ltd. (Shanghai, China), was used to transfect cells in an overnight incubation. LV3 scrambled lentiviral vector (LV3-NC; GenePharma Co., Ltd.) was used as a negative control. At 48 h after infection, the stable clones were maintained by puromycin (2-3 µg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)-resistant culturing.

Complete growth medium (RPMI-1640 with 10% FBS, 1 ml) was added to each lower chamber as a chemoattractant. Transwell inserts with a pore diameter of 8-µm were used (Millipore; Merck KGaA). Cells, suspended in serum-free medium at 5-8×10⁴ cells/ml, were seeded into the upper chamber at 400 µl/well. After an incubation of 20 h, the upper surface of the insert was wiped and cells that had migrated to the lower surface were fixed using 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min at room temperature. Cell number was counted in 6 random fields per well (magnification, ×200).

Cells were washed 3 times in PBS before the protein was extracted using radioimmunoprecipitation assay buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP-40 and 0.5% sodium deoxycholate] with protease inhibitors. The concentration of protein was detected by Bradford assay protein quantitation kit (Abcam, Cambridge, UK). Proteins (30 µg) were separated by 12% SDS-PAGE and transferred into nitrocellulose membranes. Following blocking in 5% skim milk at room temperature for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. Primary antibodies used were as follows: GAPDH (1:10,000; cat. no. KC-5G4; Kangchen Bio-tech Co., Ltd., Shanghai, China); E-cadherin (1:1,000; cat. no. sc-8426; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); Vimentin (1:200; cat. no. sc-6260; Santa Cruz Biotechnology, Inc.); CD44 (1:800; cat. no. 3570; Cell Signaling Technology, Inc., Danvers, MA, USA); Oct4 (1:500; cat. no. ab18976; Abcam); Nanog (1:200; cat. no. ab21624; Abcam); and Gli1 (1:1,000; cat. no. 2643; Cell Signaling Technology, Inc.). The membranes were then washed in Tris-buffered saline with 0.1% Tween and incubated with horseradish peroxidase-conjugated secondary antibodies [goat anti-rabbit IgG (1:2,000; cat. no. ZB-2301); and goat anti-mouse IgG (1:2,000; cat. no. ZB-2305) (all from OriGene Technologies, Inc., Beijing, China) for 1 h at room temperature. Protein bands were visualized using a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories).

For the colony-forming assay, 2×10³ cells were seeded into each well of a 6-well plate and incubated for 10-14 days. Following 3 washes in PBS, cells were fixed using 4% paraformaldehyde for 30 min at room temperature and stained with 0.1% crystal violet for 20 min at room temperature. The tumor sphere formation assay was performed by seeding 1×10⁴ cells to each well of low-adhesion 6-well plate in serum-free Dulbecco's modified Eagle/F12 medium supplemented with 20 ng/ml epidermal growth factor, 10 ng/ml basic fibroblast growth factor and 2% B27 (all Invitrogen; Thermo Fisher Scientific, Inc.). After 2 weeks, plates were analyzed for tumor sphere formation using an inverted microscope (magnification, ×200).

All statistical analysis was performed using GraphPad Prism version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). Differences between 2 groups were compared using the Student's t-test. For comparisons of ≥3 groups, one-way analysis of variance followed by Tukey's post hoc test was used. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was performed to compare the expression of miR-218 in PCa cells with that in healthy prostate epithelial cells. This revealed that the expression of miR-218 was notably downregulated in PCa cell lines, LNCaP and C4-2, compared with the normal prostate epithelial cells (BPH-1; Fig. 1A).

In order to reveal the effects of miR-218 on PCa migration, EMT and CSC properties, miR-218-overexpressing LNCaP/C4-2 cells were constructed by transfecting the cells with LV3-miR-218 lentiviral vectors. Subsequently, cells were analyzed by RT-qPCR to confirm miR-218-overexpression (Fig. 1B).

The Transwell migration assays demonstrated that the migration of LNCaP/C4-2 cells was suppressed by miR-218 overexpression (Fig. 2). Western blotting of EMT markers demonstrated that miR-218 overexpression caused a slight increase in the expression of E-cadherin and a significant decrease in the expression of vimentin (Fig. 3A). These results suggest that overexpression of miR-218 inhibits PCa cell migration and EMT.

Protein expression profiling of cancer stemness markers was performed by western blotting. The results indicate that overexpression of miR-218 downregulated the expression of CD44, Oct4 and Nanog (Fig. 3B). Colony forming assays were performed to assess the self-renewal capacity of PCa cells. It was demonstrated that the miR-218-overexpressing LNCaP cells formed fewer and smaller colonies than the control cells (Fig. 4A). Similar results were obtained from the clonogenic assay performed in C4-2 cells (Fig. 4B), indicating that miR-218 may serve a critical role in tumor growth inhibition. The tumor sphere formation assay is well established for measuring the self-renewing capability of stem cells. In these experiments, the control cells generated more tumor-spheres than the miR-218-overexpressing cells in both cell lines (Fig. 4C and D). Therefore, these results suggest that overexpression of miR-218 diminished PCa cell stemness properties.

Numerous studies have indicated that the Hedehog-Gli signaling pathway serves a critical role in cancer cell EMT occurrence and CSC generation (13–15). In the preset study, western blot analysis indicated that the expression of Gli1 was inhibited by miR-218 overexpression (Fig. 5), indicating that miR-218 suppression of Gli1 may be a mechanism for the anticancer effect of miR-218 in PCa.