Human NB SH-SY5Y cells were obtained from the American Type Culture Collection (Rockville, MA, USA), and cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA), with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific Inc., Waltham MA, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and were grown in a 5% CO₂ incubator at 37°C. The PIWIL2 inhibitor SOV was purchased from Sigma-Aldrich.

Cell viability was assessed by an MTT assay. Briefly, SH-SY5Y cells were seeded in a 96-well culture plate, and subsequently treated with different concentrations of SOV for 24, 48 or 72 h. Control cells were treated with phosphate-buffered saline (PBS) in culture medium. Following treatment, the cells were incubated with 20 µl MTT reagent (5 mg/ml; Sigma-Aldrich) for 4 h. MTT was then removed and 150 µl dimethyl sulfoxide was added, cells were then analyzed by colorimetric analysis using a multi-label plate reader (Bio-Rad, Hercules, CA, USA) at 490 nm. Experiments were conducted in triplicate, average activity rates were relative to control and standard error was calculated.

The Annexin V/fluorescein isothiocyanate (FITC) Apoptosis Detection kit (KeyGEN Biotech, Nanjing, China) was used for measuring apoptosis according to the manufacturer's protocol. Firstly, equal numbers of SH-SY5Y cells treated with PBS or SOV for 24, 48 or 72 h, were incubated with Annexin V-FITC, followed by staining of their DNA with propidium iodide (PI; Sigma-Aldrich) in the dark. Then, each sample was analyzed by fluorescence-activated cell sorting (FACSAria II; BD Biosciences, San Jose, CA, USA). The percentages of cells stained positive for Annexin V were calculated, and the means as well as the standard error were plotted.

The quantitative analysis of cell count and viability were determined using the Muse Count & Viability kit (MCH100102; Merck KGaA, Darmstadt, Germany) on the Muse Cell Analyzer (EMD Millipore, Billerica, MA, USA). The Muse Count & Viability Reagent differentially stains viable and non-viable cells based on their permeability to the two DNA binding dyes present in the reagent. First, equal numbers of SH-SY5Y cells were treated with PBS or SOV for 24, 48 or 72 h. Then a uniform cell suspension was prepared with the cell concentrations adjusted in the range of 1×10⁵–1×10⁷ cells/ml. Stained cell samples were then prepared by mixing cells with Muse Count & Viability Reagent in a sample tube. Subsequently the cells were left to take up the stain for a minimum of 5 min. The samples were then detected on Muse Cell Analyzer according to the manufacturer's protocol. The Muse Count & Viability Software Module (EMD Millipore) was then used to perform calculations automatically and display data in two dot plots.

Equal numbers of SH-SY5Y cells were plated in 10-cm dishes and treated with PBS or SOV for 24, 48 or 72 h. In total, 10⁶ cells were trypsinized (Sigma-Aldrich), fixed with 70% ethanol and incubated overnight at 4°C. Cells were then incubated in 100 µl RNase (Merck Millipore, Darmstadt, Germany) at 37°C for 30 min, followed by staining of the DNA with 400 µl PI for 30 min in the dark, and FACS analysis. The average percentages of cells in G0/G1, S or G2/M phases of the cell cycle were quantified and standard error was calculated for three experiments.

Total RNA was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) and quantified using NanoDrop 1000 (NanoDrop, Wilmington, DE, USA). Then the RNA was reverse transcribed into cDNA using PrimeScript RT reagent kit with gDNA Eraser (Takara Bio Inc., Otsu, Japan). RT-qPCR was performed using SYBR Premix Ex Taq™ II (Takara Bio Inc.) on a ABI 7500 Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific Inc.). A total of 520 ng of cDNA was obtained. The following primer sequences were used: Forward: 5′-CGGAATGACTGTGTGCTGGA-3 and reverse: 5′-GGTGATAACAATATTGCCAACCAGA-3′ for human PIWIL2; and forward: 5′-TGGCACCCAGCACAATGAA-3′ and reverse: 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′ for human β-actin. Primers were obtained from Takara Biotechnology Co., Ltd. (Dalian China) The PCR amplification was conducted under the following conditions: 95°C for 30 sec; 95°C for 5 sec, and 60°C for 34 sec for 40 cycles. The relative changes in gene expression data were analyzed by the 2^−ΔΔCt method. β-actin was used as an internal control. Triplicates were run for each sample in three independent experiments.

Equal numbers of SH-SY5Y cells were plated on 10-cm plates and treated with PBS or SOV for 24, 48 or 72 h. Then the cells were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) and protein concentration was determined by the Bradford method using a Bicinchoninic Acid Protein Assay kit (cat. no. P0012; Beyotime Institute of Biotechnology) (21,22). Equal amounts of protein (40 µg) were separated by sodium dodecyl sufate polyacrylamide gel electrophoresis and then transferred onto polyvinylidine difluoride membranes (EMD Millipore). The blotted membranes were blocked with 5% non-fat dry milk (w/v) in Tris-buffered saline with 0.1% Tween 20, and then incubated at 4°C overnight with rabbit monoclonal anti-PIWIL2 (Bioss Inc., Woburn, MA, USA; cat. no. bs-3817R) and anti-Bcl-2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; cat. no. sc-492) antibodies. Specific antibody binding was detected using polyclonal horseradish peroxidase-conjugated goat anti-rabbit antibodies (cat. no. A0208; Beyotime Institute of Biotechnloogy; 1:1,000) and visualized with an enhanced chemiluminescence reagent (cat. no. sc-2048; Santa Cruz Biotechnology Inc.), according to the manufacturer's protocol. Goat polyclonal IgG antibodies against β-actin (cat. no. sc-1616; Santa Cruz Biotechnology, Inc.; 1:200) was used to evaluate protein loading in each lane. Quantification was performed using Image J software version 1.44 (National Institutes of Health, Bethesda, MA, USA)

Each experiment was repeated three times. All quantitative variables were analyzed using one way analysis of variance followed by Tukey's post-hoc test using SPSS software, version 16.0 (SPSS, Inc., Chicago, IL, USA). The results are presented as the mean ± standard deviation, and the standard deviation were presented as error bars in the figures. P<0.05 was considered to indicate a statistically significant difference.

SOV has been described as a potent small molecule inhibitor of PIWIL2 and could induce cell apoptosis and inhibit autophagy in human hepatocellular carcinoma (HCC) cells, in vitro and in vivo (18). To elucidate the role of SOV in NB, it was investigated how different concentrations of SOV affect cell proliferation in SH-SY5Y cells with different concentrations. SH-SY5Y cells showed reduction in cell proliferation after 24 h of treatment with 5 µM SOV, with a maximum reduction at 72 h (Fig. 1). This anti-proliferative effect occurred in a dose- and time-dependent manner at 10 and 50 µM at 24, 48 and 72 h. These results indicate that SOV attenuates NB cell proliferation. Thus 5 µM SOV was selected for use in further assays.

The early apoptotic cells were stained with Annexin V, and are observed in the right lower quadrant (Fig. 2A). The results showed that the apoptotic rates in the SOV group were 2.70±0.20, 3.53±1.50 and 7.93±1.07% respectively, at 24, 48 and 72 h, which were significantly higher than those in control group (P<0.05, Fig. 2B). In addition, the apoptotic rates increased with time following SOV treatment, and reached a climax at 72 h (Fig. 2B, P<0.01). These results indicated that apoptosis was induced by SOV in SH-SY5Y cells.

Viable cells are located in the top left quadrant and dead cells are located in the lower right quadrant (Fig. 3A). After SOV treatment, the average percentages of viable cells were 57.59±8.72, 59.47±5.36 and 60.57±14.72% at 24, 48 and 72 h respectively, while those in control group were 71.17±1.21, 70.9±0.91 and 80.98±2.30%, respectively (Fig. 3B, P<0.05). This suggested that SOV could reduce the percentage of viable cells, particularly at 72 h where they were reduced by ~25.2% (Fig. 3B, P<0.05).

As shown in Fig. 4A, the number of SH-SY5Y cells in the G0/G1 phase decreased, while those in the S and G2/M phase increased following SOV treatment. At 24 h after treatment, 45.48% of the SOV-treated SH-SY5Y cells were in the G0/G1 phase, 40.10% at S and 14.41% at G2/M (Fig. 4B, P<0.05). By comparison, 73.15% of the PBS-treated SH-SY5Y cells were at G0/G1, 21.26% at S and 5.58% at G2/M (Fig. 4B, P<0.05). This trend was continued at later time points (Fig. 4B). This suggested that PIWIL2 is key in cell cycle regulation and that SOV induces accumulation of SH-SY5Y cells at the G2/M and S phase of the cell cycle.

RT-qPCR showed that the relative expression of PIWIL2 mRNA was 0.45±0.02 following SOV treatment, which was lower than that in the control group (Fig. 5, P<0.05). The results indicated that SOV treatment suppressed the expression of PIWIL2 mRNA to a certain extent.

It has been reported that knocking out the PIWIL2 gene in SW620 and SW480 colon cancer cell lines inhibited cell proliferation, invasion and metastasis in vitro, and tumorigenicity in vivo (17). Western blot analysis was performed to determine whether PIWIL2 inhibition induces apoptosis in SH-SY5Y cells and whether Bcl-2 is involved. PIWIL2 expression in SH-SY5Y cells was inhibited by 5 µM SOV. Following inhibition of PIWIL2 in SH-SY5Y cells, protein levels of PIWIL2 and Bcl-2 were reduced (Fig. 6, P<0.05). This indicates that SOV may induce apoptosis in SH-SY5Y cells in part by reducing the expression of the anti-apoptotic protein Bcl-2.