The PC3 human prostate cancer cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in Gibco™ RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine (both Sigma-Aldrich, St. Louis, MO, USA) at 37°C in a humidified atmosphere containing 5% CO₂. Plasmids encoding LSD1 small interfering (si)RNA or mock vehicle pCMV-G&NR-U6-shRNA (GeneChem Co., Ltd., Shanghai, China) were transfected into the PC3 cells in 6-well plates using a lentiviral vector (JRDUN Biotechnology, Shanghai, China) and Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. Three sequences of the LSD1 were used, as follows: Short hairpin (sh)LSD1-1, 5′-ACGAAAGTGTCTCCGTTGA-3′; shLSD1-2, 5′-CCGACATGGCTTTCTCTTT-3′; and shLSD1-3, 5′-TCGACAGTGACCCCTTATA-3′. The cells were split twice weekly, and cells in the logarithmic growth phase were used for subsequent experiments.

Total RNA was extracted from the cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and the mRNA was reverse transcribed into cDNA using the TIANScript RT kit (Tiangen Biotech. Co. Ltd., Shanghai, China). Subsequently, qPCR was conducted using the SYBR Green PCR Master mix (Thermo Fisher Scientific, Inc.) and the ABI 7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), in which glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as the reference gene. The following primers were used: LSD1, forward (F) 5′-AAGCAGGAGGACTTCAAGAC-3′, reverse (R) 5′-GCAGTGTGCGGTTTCTAATG-3′; GAPDH, F 5′-CACCCACTCCTCCACCTTTG-3′ and R 5′-CCACCACCCTGTTGCTGTAG-3′ (Generay Biotech Co., Ltd., Shanghai, China). The PCR cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. RT-qPCR data were analyzed with SDS 2.3 software. Each experiment was repeated three times and the relative mRNA expression levels of LSD1 were calculated using the 2^−ΔΔCq method (19).

The viability of the PC3 cells was measured using a CCK-8 kit (Boster Biological Technology, Ltd., Wuhan, China). The cells were seeded into a 96-well microplate at a density of 5×10³/well and incubated for 24 h. The peripheral wells of the microplate contained phosphate-buffered saline (PBS) only. The cells were divided into four groups: Mock vehicle group, LSD1 siRNA group, mock vehicle + DDP group or LSD1 siRNA + DDP group, according to the experimental design. The cells were treated with 5 µg/ml DDP (Beyotime Institute of Biotechnology, Haimen, China), after which the cells were incubated for a further 24 h prior to the assay. A total of 10 µl CCK8 solution was added to each well containing PC3 cells, and the cells were incubated for 0, 24, 48 or 72 h. Finally, the absorbance of each well was measured at 490 nm using a Gemini XPS microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

The PC3 cells (5×10⁵ cells/ml) were inoculated into 6-well plates. Each group comprised three double wells on the plate. Following a 24 h incubation, the groups were generated by addition of the appropriate reagents to the cells, and the cells were incubated for a further 24 h. The flow cytometric analysis was performed according to the protocol of the Annexin V-Fluorescein Isothiocyanate Apoptosis Detection kit (Abcam, Cambridge, UK). The apoptotic rates were analyzed immediately using a FACSCalibur™ flow cytometer (BD Biosciences, San Jose, CA, USA).

Following the removal of the culture medium from each group, the cells were digested with trypsin and diluted to 1×10⁵/ml in serum-free Dulbecco's Modified Eagle's Medium containing 1% FBS (GE Healthcare Life Sciences, Logan, UT, USA). A total of 800 µl culture medium containing 10% FBS was added to the coated lower chambers of a Transwell system. Matrigel (BD Biosciences) was coated onto the upper chambers of the Transwell system, after which 200 µl cell suspension was added to the upper chambers at a density of 5×10⁴/well. The plate was cultured for 24 h, after which the cells on the upper layer were removed. The cells that had migrated to the lower layer were washed with PBS, fixed with methanol and stained with 1% crystal violet. The invasive cells were counted in five fields for each sample under an inverted microscope (BX51; Olympus Corporation, Tokyo, Japan), and the results were averaged. The experiments were repeated three times.

Total protein was extracted from the cell samples using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology), and was quantified using the Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). Subsequently, equal volumes of protein (30 µg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting onto a nitrocellulose membrane (EMD Millipore, Billerica, MA, USA) using an electrophoretic transfer cell (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked with 5% skimmed milk, followed by incubation overnight at 4°C with the following primary antibodies: Monoclonal anti-E-cadherin (#14472; 1:1,000), polyclonal anti-cyclo-oxygenase-2 (COX-2; #4842; 1:1,000), monoclonal anti-Smad2/3 (#8685; 1:1,000), monoclonal anti-phosphorylated (p)-Smad2/3 (#8828; 1:1,000), monoclonal anti-cyclin D (#2978S; 1:1,000), polyclonal anti-p-Akt (#9271; 1:1,000), polyclonal anti-Akt (#9272; 1:1,000), monoclonal anti-extracellular signal-regulated kinase (ERK; #4695; 1:1,000), monoclonal anti-p-ERK (#4376; 1:1,000) and monoclonal anti-GAPDH (#5174; 1:1,500) from Cell Signaling Technology, Inc. (Danvers, MA, USA), and polyclonal anti-transforming growth factor-β1 (TGF-β1; ab92486; 1:800) and anti-vascular endothelial growth factor (VEGF; ab46154; 1:1,000) from Abcam. Subsequently, the blots were washed three times with PBS and incubated with horseradish peroxidase-conjugated goat anti-mouse (A0216; 1:1,000) or goat anti-rabbit (A0208; 1:1,000; both Beyotime Institute of Biotechnology) secondary antibodies for 1 h at room temperature. The bands were detected by reaction with enhanced chemiluminescence detection system reagents (EMD Millipore) and exposure to X-ray film (Kodak, Rochester, NY, USA), which was subsequently developed and used to capture photographic images. GADPH was used to normalize the protein expression. Band intensities were analyzed using ImageJ 1.49 software (https://imagej.nih.gov/ij/).

All data are presented as the mean ± standard deviation. The data were evaluated using the Prism 5.0 statistical software package (GraphPad Software Inc., San Diego, CA, USA). The two-tailed Student's t-test was used to evaluate statistical differences between two groups. P<0.05 was considered to indicate a statistically significant difference.

The present study hypothesized that LSD1 may have an important role in PC3 cells, which has not been previously investigated. Lentiviral-mediated RNA interference technology was used to establish a stably transfected LSD1 knockdown PC3 cell line, and the CCK-8 colorimetric assay was subsequently used to determine cell proliferation. As shown in Fig. 1A, the mRNA expression levels of LSD1 in the knockdown group were decreased to <60% of the control levels (P<0.01), whereas the mRNA expression levels of LSD1 in the mock group (i.e. control cells which were transfected with an irrelevant interference sequence) exhibited no significant changes. All of the groups contained similar cell numbers at the 0 h time point; however, proliferation of the LSD1 knockdown PC3 cells was significantly decreased after 24, 48 and 72 h, as compared with the mock group (P<0.01; Fig. 1B). Furthermore, compared with the mock group, DDP (at a concentration of 5 µg/ml) exerted a marked inhibitory effect on PC3 cell proliferation. Notably, the LSD1 knockdown + DDP group demonstrated a more marked inhibition on PC3 cell proliferation, as compared with the DDP or LSD1 knockdown groups (P<0.05). These findings indicate that the proliferative capability of the PC3 cells was decreased following LSD1 knockdown, thus suggesting that LSD1 may contribute to the proliferation of PC3 cells, and that siRNA interference may result in reduced cell growth. Furthermore, DDP had similar effects to LSD1 siRNA, and a combination of LSD1 knockdown and DDP treatment produced a synergistic effect.

The results of the present study revealed that cell proliferation was inhibited when LSD1 was knocked down and the cells were co-treated with DDP, as compared with the mock group. Since the Akt and ERK signal transduction pathways are reported to modulate cell proliferation and tumorigenicity (20,21), the protein expression levels of p-Akt and p-ERK were subsequently investigated. Furthermore, cyclin D has a crucial role in the Akt signal pathway as a regulatory protein of the cell cycle (22), and therefore the protein expression levels of cyclin D were also investigated. The protein expression levels of p-Akt, p-ERK and cyclin D1 were downregulated in the treatment groups (LSD1, mock + DDP, LSD1 + DDP), as compared with the mock group (Fig. 1C and D). These results suggest that LSD1 RNA interference and treatment with DDP may inhibit PC3 cell proliferation via regulation of the Akt and ERK signal pathways.

In addition to regulating PC3 cell growth, LSD1 may be involved in regulating cell apoptosis. As shown in Fig. 2A and B the proportion of PC3 cells in the early apoptotic phase was significantly increased following LSD1 knockdown, as compared with the mock group. Furthermore, it was revealed that DDP contributes to the increased levels of apoptosis in the PC3 cells, and that treatment with DDP exerted a synergistic action on PC3 cell apoptosis when combined with LSD1 siRNA. These results suggest that a combination of LSD1 knockdown and DDP treatment may contribute to the increased percentage of PC3 cells in the early apoptotic stage.

Invasive capability is essential for the malignant progression of tumors. As shown in Fig. 3A and B, the invasive capability of the PC3 cells was decreased following LSD1 knockdown or treatment with DDP, as compared with the mock group. Furthermore, a combination of LSD1 knockdown and DDP treatment exerted a synergistic effect on the decline in invasive ability. These results suggest that LSD1 may have a crucial role in the invasion of PC3 cells.

TGF-β1 is a cytokine peptide, which is associated with various biological roles in cancer (23). The Smad proteins act as substrates of the TGF-β1 receptor, and their activation by phosphorylation propagates the TGF-β1 signal transduction. In particular, the TGF-β1/Smad2/3 signaling pathway is involved in mechanisms underlying the invasion and metastasis of prostate cancer cells, a process which is also regulated by tumor angiogenesis, the host immune system, the cells themselves and the surrounding matrix microenvironment (24–26). Consequently, the protein expression levels of TGF-β1, p-Smad2/3, VEGF, COX-2 and E-cadherin, all of which are associated with the TGF-β1/Smad2/3 signal transduction pathway, were investigated. The results revealed that the expression levels of TGF-β1, p-Smad2/3, VEGF and COX-2 in the DDP-treated cells were downregulated, whereas the protein expression levels of E-cadherin were upregulated. Furthermore, the combination of LSD1 knockdown and treatment with DDP exerted a synergistic effect, as compared with either treatment taken in isolation or with the mock cells (Fig. 3C–E).