MCF-7 and T47D human breast cancer cells (The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences) were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.), 100 U/ml penicillin G and 0.1 mg/ml streptomycin and placed at 37°C in a humidified incubator containing 5% CO₂. The overexpression plasmid of SMYD3 was a gift from Professor Philip Tucker (Institute for Cellular and Molecular Biology, University of Texas, Austin, TX, USA), and the empty vector was used as a control. The small interfering (si) RNAs were synthesized by Guangzhou RiboBio Co., Ltd. The sequences of these siRNAs were as follows: siRNA targeting SMYD3 (si-SMYD3), sense, 5′-CAAGGAUGCUGAUAUGCUAdTdT-3′ and antisense, 3′-dTdTGUUCCUACGACUAUACGAU-5′. The sequence of the control siRNA was 5′-ACTGTTCTATGACTTGTCGTGAATA-3′. Transient transfection was performed using TurboFect reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, 1 µg plasmid or siRNA were transfected into MCF-7 or T47D cells with 2 µl TurboFect reagent. Cells were collected 24 or 48 h later for subsequent testing.

Total RNA was isolated from MCF-7 and T47D cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) under RNase-free conditions. After quantification of RNA using a photometer, cDNA was synthesized using M-MLV reverse transcriptase (Invitrogen, Thermo Fisher Scientific, Inc.) from 2 µg total RNA, according to the manufacturer's instructions.

For semi-qPCR, primers specific for the cDNAs of the SMYD3 gene and the constitutive GAPDH gene were used (Genewiz Inc.). For SMYD3, the forward primer positions were 647–670 (5′-CCCAGTATGTCTTTGCTGAATCAC-3′) and the reverse primer positions were 935–956 (5′-ACTTCCAGTGCGCCTTCAGCTC-3′). For GAPDH, the forward primer positions were 217–236 (5′-ATTCAACGGCACAGTCAAGG-3′) and the reverse primer positions were 411–429 (5′-GCAGAAGGGGCGGAGATGA-3′). The PCR reactions were as follows: 95°C for 5 min, then 30 cycles at 95°C for 1 min, at 56°C for 1 min, and at 72°C for 30 sec; extension was carried out at 72°C for 10 min. PCR products were electrophoretically separated by 1.5% agarose gels and expression observed using ethidium bromide staining. The densities of the bands were analyzed using Gel/Chemi Doc (Bio-Rad Laboratories, Inc.) and quantified using Quantity One software v 4.6.6 (Bio-Rad Laboratories, Inc.).

qPCR was performed using Fast SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific Inc.) and detected using an ABI Step One system (Thermo Fisher Scientific, Inc.). The sequences of the primers (Invitrogen; Thermo Fisher Scientific, Inc.) were as follows: GAPDH forward, 5′-ATTCAACGGCACAGTCAAGG-3′ and reverse, 5′-GCAGAAGGGGCGGAGATGA-3′; SMYD3 forward, 5′-CCCAGTATCTCTTTGCTCAATCAC-3′ and reverse, 5′-ACTTCCAGTGTGCCTTCAGTTC-3′; miR-124 forward, 5′-ACACTCCAGCTGGGTAAGGCACGCGG-3′ and reverse, 5′-TGGTGTCGTGGAGTCG-3′. qPCR was performed as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 10 sec, 65°C for 30 sec and 72°C for 30 sec. The 2^−ΔΔCq method was used to calculate relative transcription levels and all experiments were repeated at least three times (24).

A total of 48 h after transfection, MCF-7 and T47D cells were washed twice with ice-cold PBS and lysed at 4°C with RIPA lysis buffer containing protease and phosphatase inhibitors (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min. The lysates were centrifuged at 17,000 × g for 10 min, the supernatants were quantified using the BCA protein concentration assay kit (Beyotime Institute of Biotechnology) and mixed with SDS sample buffer. Subsequently, proteins (50 µg/lane) were separated using 12% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked using 5% non-fat dry milk dissolved in PBS for 90 min at 20°C. Membranes were then incubated with primary antibodies against SMYD3 (rabbit anti-human monoclonal antibody; 1:1,000; cat. no. ab187149; Abcam) and GAPDH (mouse anti-human; dilution; 1:5,000; cat. no. sc-365402; Santa Cruz) at 4°C overnight. Subsequently, membranes were incubated with infrared fluorescent goat anti-rabbit (1:10,000; cat. no. 926-68071; LI-COR Biosciences;) and goat anti-mouse (1:10,000; cat. no. 926-32210; LI-COR Biosciences;) secondary antibodies for 2 h in the dark at room temperature. Signals were visualized using an Odyssey Imaging System (LI-COR Biosciences).

The SMYD3 3′-untranslated region (3′UTR) was subcloned into pGL3 Basic vector (Promega Corporation). MCF-7 or T47D cells were seeded into 24-well plates at a density of 1×10⁸ cells/ml and then treated with different concentrations of cisplatin (0, 2, 4, 8, 16, 32 or 64 µM) for 24 h and transfected with pGL3-SMYD3 3′UTR and pEGFP-C3 plasmids using TurboFect reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The luciferase activity was measured using the Luciferase assay system (Promega Corporation) according to the manufacturer's instructions. The values were normalized to the signal of enhanced green fluorescent protein and the 0 µM cisplatin group was used as control group (25).

MCF-7 and T47D cells were seeded into 96-well plates at the density of 5×10⁴ cells/ml. After serum-free medium starvation for 24 h, cells were transfected with siRNA or overexpression SMYD3 and corresponding controls as aforementioned. Cells transfected with overexpression or small interference SMYD3 and co-treated with cisplatin for 24, 48 and 72 h were incubated with 5 mg/ml MTT (Beijing Solarbio Science & Technology Co., Ltd.) at 37°C for 4 h. Subsequently, medium was removed, 150 µl DMSO was added in each well to dissolve formazan crystals and optical density (OD) was measured at 570 nm using a Synergy4 microplate reader (BioTek Instruments, Inc.; Agilent Technologies, Inc.). Cell viability was calculated as follows: Cell viability (%)=[OD (treated)-OD (blank)]/[OD (untreated)-OD (blank)] ×100. Each sample was examined in duplicate, and the experiment was repeated three times.

Following transfection with siRNA or treatment with 0, 2, 4, 8, 16 and 32 µM cisplatin at 37°C for 24 h, cells were seeded in a 6-well plate at a density of 1,000 cells/well. Cells were subsequently cultured for 10 days at 37°C to obtain visible colonies. Cells were washed twice with PBS, fixed with methanol for 15 min at room temperature and stained with Giemsa for 30 min at room temperature. An inverted fluorescence microscope, ECLIPSE TE2000 (20×) was used to count colonies containing at least 50 cells (Nikon Corporation).

Previous studies demonstrated that cisplatin can promote apoptosis by reducing mitochondrial membrane potential (26,27). In the present study, MCF-7 cells were treated with 0 or 8 µM cisplatin and/or siRNA targeting SMYD3 mRNA for 48 h at 37°C. Then cells were harvested with trypsin and resuspended in medium at a density of 1×10⁶ cells/ml. Subsequently, cells were mixed with 50 µg/ml rhodamine 123 dye solution (Sigma-Aldrich; Merck KGaA), which is a positively charged pigment that can bind to mitochondria with a high negative membrane potential (28). After being incubated at 37°C and 5% CO₂ for 30 min. Cells were collected by centrifugation at 1,700 × g for 10 min at room temperature, washed twice with PBS and analyzed in the Accuri C6 flow cytometer (Becton Dickinson Biosciences, USA) using FlowJo v 10.2 (FlowJo LLC).

Data are presented as the means ± standard deviation (mean ± SD). Significant differences were evaluated using two-way ANOVA. Statistical analyses were performed using GraphPad Prism version 5.00 (GraphPad Software, Inc.). All experiments were carried out at least three times. P<0.05 was considered to indicate a statistically significant difference.

MTT assays were performed to assess the effect of cisplatin on MCF-7 and T47D cell viability after 24 and 48 h treatment. As presented in Fig. 1A, following cisplatin treatment, viability of T47D cells was lower compared with MCF-7 cells. This indicates that MCF-7 cells may be more sensitive to cisplatin. Furthermore, the expression of SMYD3 in these two cell lines was further investigated by RT-qPCR and western blotting. The results demonstrated that both mRNA and protein level of SMYD3 in the T74D cells were higher compared with MCF-7 cells (Fig. 1C). To quantitatively assess the effects of cisplatin on these two cell lines, the half maximal inhibitory concentration (IC₅₀) values of cisplatin were calculated. As presented in Fig. 1B, following 24 h of cisplatin treatment, the observed IC₅₀ values for MCF-7 and T47D cells were 62.13±3.58 and 342.76±17.66 µM, respectively. These results indicated that the differential expression of SMYD3 observed may be associated with the different cisplatin sensitivities of these tumor cell lines.

SMYD3 is often highly expressed in cancer tissues and is closely associated with malignancy and poor prognosis of patients (29,30). However, the association between SMYD3 expression, chemotherapy and its outcomes remains unclear. In the present study, SMYD3 was significantly knocked down by siRNA transfection in MCF-7 and T47D cell lines (Fig. 2A), and MTT and colony-formation assays were performed to assess the cisplatin sensitivity in these two cell lines. The results demonstrated that SMYD3 knockdown significantly reduced the cell viability following cisplatin treatment (Fig. 2B and C), suggesting that SMYD3 may be involved in the development of cell resistance to cisplatin. To verify this hypothesis, further colony-formation assays were performed using MCF-7 cells (Fig. 2D) and the sensitivity of these cells to cisplatin was enhanced after SMYD3 knockdown. In addition, to investigate whether SMYD3 can affect the effect of cisplatin on the mitochondrial membrane potential of cells, rhodamine 123 staining was performed. As presented in Fig. 2E, SMYD3 knockdown decreased the mitochondrial membrane potential, which was enhanced in the presence of cisplatin. These results demonstrated that SMYD3 knockdown and cisplatin treatment may cooperatively promote the loss of the mitochondrial membrane potential in cancer cells.

MCF-7 cells were successfully transfected with the overexpression plasmid of SMYD3 or a control empty plasmid. (Fig. 3A). Following transfection for 24 h, SMYD3-overexpressing cells exhibited increased cell viability and increased tolerance to cisplatin compared with control (Fig. 3B). Similar results were also observed following transfection of cells with the SMYD3 overexpression plasmid for 48 h (Fig. 3B). The results of the colony-formation assay also demonstrated that overexpression of SMYD3 could significantly increase the tolerance of cells to cisplatin (Fig. 3C).

The aforementioned results demonstrated that cell sensitivity to cisplatin may be associated with SMYD3 expression. To determine the underlying mechanisms of cisplatin-mediated cytotoxicity, MCF-7 and T47D cells were treated with cisplatin at different concentrations for 24 or 48 h, and SMYD3 expression level was assessed by RT-PCR and western blotting. Following treatment with cisplatin for 24 and 48 h, SMYD3 mRNA level was significantly downregulated, suggesting that cisplatin can inhibit the transcription of SMYD3 (Fig. 4A and B). These results of western blotting analysis also demonstrated that SMYD3 expression was decreased following cisplatin treatment in a concentration-dependent manner (Fig. 4C).

Since miRNAs can combine with the 3′UTRs of mRNAs to inhibit transcription (31). In the present study, a luciferase reporter assay was used to test whether cisplatin-mediated inhibition of SMYD3 expression may occur via the regulation of a specific miRNA that mediates SMYD3 transcription. This assay assessed the transcriptional activity of SMYD3 3′UTR following treatment with different concentrations of cisplatin (Fig. 5). Following cisplatin treatment, the luciferase activity of the SMYD3 3′UTR was significantly downregulated (Fig. 5A), suggesting that cisplatin may inhibit SMYD3 expression by upregulating the transcription of a miRNA that could promote the degradation of SMYD3 mRNA.

As previous studies reported that miR-124 is an upstream regulator of SMYD3 (20,32), the present study hypothesized that miR-124 may be involved in the inhibitory effect of cisplatin on the expression of SMYD3. To verify this hypothesis, MCF-7 and T47D cells were treated for 24 h with different concentrations of cisplatin and the level of miR-124 was assessed by RT-qPCR. For both cell lines, miR-124 level was significantly increased following cisplatin treatment in concentration-dependent manner in MCF-7 and T47D cell lines, respectively (Fig. 5B), suggesting that cisplatin may inhibit SMYD3 expression by upregulating miR-124.