All patients with NB (aged between 7 months and 8 years) were clinically and histopathologically diagnosed at Beijing Children's Hospital between May 2015 and December 2016 based on the International Neuroblastoma Staging System (INSS) for clinical staging of NB (22). The present study was approved by the Ethics Committees of Beijing Children's Hospital. A total of 40 surgical specimens were immediately snap-frozen in liquid nitrogen prior to total RNA extraction.

The NB cell line SH-SY5Y (#CRL-2266; MYCN non-amplified) was obtained from the American Type Culture Collection (ATCC); this cell line is widely used in mechanistic and drug development studies regarding NB (23,24). Cells were cultured in Dulbecco's modified Eagle's medium (Corning, Inc.) supplemented with 10% fetal bovine serum (FBS; Corning, Inc.) in a humidified incubator containing 5% CO₂ at 37°C. According to the manufacturer's protocol, synthetic small interfering (si)RNAs were transfected into cells at ~50% confluence using the Lipofectamine RNAiMAX kit (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were further analyzed 8 h post-transfection.

SH-SY5Y cells in the exponential growth phase were seeded for 24 h and were then transfected with 100 nM siRNA at room temperature using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.). siRNA oligonucleotides were synthesized by Sangon Biotech Co., Ltd., as follows: SNHG16 (siRNA1-SNHG16, 5′-CAGCAGUUGAGGGUUUGCUGUGUAUdTdT-3′, siRNA2-SNHG16, 5′-GGACAACCUAGCUGUUGAAdTdT-3′); non-targeting control (5′-UUCUCCGAACGUGUCACGUTT-3′). Cells were returned to the incubator and were refreshed with normal medium 8 h post-transfection, and further experiments were performed at scheduled times.

A total of 3×10⁵ SH-SY5Y cells/well were seeded in 6-well plates at ~50% confluence and transfected with siRNA for 72 h. Tumor tissues and cells were homogenized in TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and total RNA was extracted using the Direct-zol™ RNA Miniprep kit (Zymo Research Corp.). The RevertAid™ H Minus First Strand cDNA Synthesis kit (Invitrogen) was used for RT, and cDNA templates were amplified with the SYBR Green Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). Both RNA extraction and RT procedures were performed according to the manufacturers' protocols. qPCR thermal cycling was set as follows: Initial denaturation at 95°C for 10 min; 40 cycles at 95°C for 15 sec and 60°C for 60 sec; followed by melt curve at 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec. For RNA expression detection, GAPDH was used as a reference gene. The primer sequences were as follows: SNHG16, forward 5′-CAGTCAGCCTCAGTTTCCAA-3′, reverse 5′-AGGCAGGGCTGTGCTGAT-3′; and GAPDH, forward 5′-CGAGTCAACGGATTTGGTGGTAT-3′ and reverse 5′-AGCCTTCTCCATGGTGAAGAC-3′. The relative fold-change in mRNA expression was calculated using the 2^−ΔΔCq method (25).

Cell proliferation was measured by xCELLigence real-time cell analysis (RTCA) (ACEA Biosciences, Inc.). Briefly, a total of 4 ×10³ SH-SY5Y cells were cultured in an adaptive E-plate (ACEA Biosciences, Inc.) and transfected with siRNA. The E-plate was then incubated at 37°C within the RTCA Station inside the incubator, and device-defined cell index values were recorded every 20 min for 72 h. Increases in cell numbers altered the baseline impedance, which was monitored by gold micro-electrodes located at the bottom of the E-plate. Data analysis was performed using the RTCA Control Unit and preinstalled RTCA software 2.0 (ACEA Biosciences, Inc.).

A total of 2×10³ SH-SY5Y cells/well were seeded in 6-well plates and transfected with siRNA. After 14 days, cells were fixed with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) for a further 10 min at room temperature. Images were captured using a light microscope (IX73; Olympus Corporation), in order to observe cell colony formation. Colonies containing >50 cells were counted and recorded for statistical analysis. Independent assays were conducted three times.

A total of 3×10⁵ SH-SY5Y cells/well were seeded in 6-well plates at ~50% confluence, and a 1-ml micropipette tip was used to scratch the surface of the plate to create a 'wound'. After gentle washing with phosphate-buffered saline (PBS), the attached cells were transfected with siRNA. Wound images were captured under a light microscope (IX73; Olympus Corporation) at 0, 24, 48 and 72 h. The wound width was measured and analyzed by AlphaView SA 3.4.0 software (ProteinSimple).

Transwell chambers (pore size, 8 μm; Costar; Corning, Inc.) were used to conduct a migration assay. A total of 250 μl serum-free medium containing 4×10⁴ cells was added into the upper chamber, whereas 500 μl complete medium was added into the bottom chamber. After 24 h at 37°C, the cells on the upper chamber were discarded and the cells that had migrated to the lower surfaces of the filters were fixed with 4% para-formaldehyde for 10 min at room temperature. Images of the cells were captured using a light microscope (IX73; Olympus Corporation) following further staining with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) at room temperature for 10 min.

A total of 3×10⁵ SH-SY5Y cells/well were cultured in 6-well plates; 72 h post-transfection, cells were trypsinized, centrifuged at 110 × g for 5 min, and resuspended in ice cold ethanol (70%) at 4°C overnight. PBS containing 2% FBS was added to spin down the cells at 440 × g for 5 min. The cell pellet was then incubated in PBS containing 2% FBS, 10 μl 1 mg/ml propidium iodide solution and 2 μl 10 mg/ml RNAseA (Tiangen Biotech Co., Ltd.) for 30 min at 37°C in the dark. Cell cycle progression was assessed using a flow cytometer and CellQuest Pro 6.1 software (BD Biosciences); ≥1×10⁴ cells were analyzed for each sample.

AO and EB staining (Nanjing KeyGen Biotech Co., Ltd.) was used to visualize nuclear alterations characteristic of apoptosis. AO is a vital dye that stains live and dead cells, whereas EB only stains cells that have lost membrane integrity, thus indicating apoptosis. Live cells appear uniformly green, whereas apoptotic cells will incorporate EB and therefore will be stained red-orange with condensed nuclei (26). A total of 3×10⁵ SH-SY5Y cells/well were cultured in 6-well plates, transfected with siRNA for 72 h, and stained with AO (10 μg/ml) and EB (10 μg/ml) for 30 min at room temperature. Cellular apoptosis was subsequently viewed and images were captured under a fluorescence microscope (Olympus Corporation).

Caspase-3/7 activation was measured using the Caspase-Glo 3/7 assay (Promega Corporation) according to the manufacturer's protocol. Briefly, a total of 1×10⁴ cells/well were seeded into 96-well plates, transfected with siRNA, and cultured for 72 h. Subsequently, 100 μl Caspase-Glo 3/7 reagent was added to each well and the well contents were agitated on a plate shaker. Finally, samples were incubated at room temperature for 2 h and luminescence was measured using a SpectraMax Microplate Luminometer (Molecular Devices, LLC).

Potential RBPs that bind SNHG16 (27) were systematically identified using starBase v2.0 software (starbase.sysu.edu.cn). The Cytoscape plug-in ClueGO was then used to identify Gene Ontology (GO) terms and interpret functions enriched for the predicted RBPs (28). Statistical parameters were set as follows: Right-sided hypergeometric test, P<0.05 with Benjamini-Hochberg correction; GO levels, 6-14; Kappa score threshold, 0.4.

Publically available Gene Expression Omnibus (GEO) datasets (www.ncbi.nlm.nih.gov/geo) GSE62564 (29-32) and GSE16237 (33) were downloaded for SNHG16 expression analysis. Kaplan-Meier survival analysis and the log-rank test were performed based on survival times collected from the GSE62564 dataset. All data were analyzed using SPSS 19.0 software (IBM Corp.), and graphs were generated using GraphPad Prism 5.0 (GraphPad Software, Inc.). All results are expressed as the means ± standard deviations. Multiple comparisons were assessed by one-way analysis of variance followed by Bonferroni post hoc test. Student's t-test was performed to analyze differences between two groups. P<0.05 was considered to indicate a statistically significant difference.

To explore the relationship between SNHG16 expression and the pathophysiological features of patients with NB, the GEO dataset GSE62564 (498 samples) was analyzed by stratification analysis based on INSS stage, risk group and MYCN status. SNHG16 was expressed at significantly higher levels in stage 4 tumors compared with in tumors at other stages (Fig. 1A). In addition, it was upregulated in high-risk NB and MYCN amplification subtypes compared with in low-risk NB (Fig. 1B) and MYCN non-amplification subtypes (Fig. 1C). Analysis of another independent dataset, GSE16237 (51 samples), confirmed these results (Fig. 1D and E). Analysis of NB tissue samples (Table I) revealed that SNHG16 expression was increased alongside clinical staging of NB tumor progression (Fig. 1F) and in MYCN-amplified NB (Fig. 1G). These findings validated that SNHG16 expression was positively associated with NB progression.

To explore the prognostic value of SNHG16, Kaplan-Meier survival analysis was conducted based on GSE62564 data. Patients with low or high expression of SNHG16 were grouped according to RNA sequencing data. Patients with high expression had worse event-free survival (P<0.0001) and overall survival (P<0.0001) than those with low expression (Fig. 2A and B), thus indicating that SNHG16 may be a prognostic marker for patients with NB.

The gene knockdown efficiency of siRNAs was validated by RT-qPCR. siRNA1-SNHG16 and siRNA2-SNHG16 effectively silenced SNHG16 expression (Fig. 3A). The real-time cell proliferation assay revealed that cell growth in the SNHG16 silencing group was significantly inhibited (Fig. 3B). This was confirmed by the colony formation assay, which demonstrated that SNHG16 silencing markedly reduced colony formation in SH-SY5Y cells (Fig. 3C and D). These findings indicated that SNHG16 may contribute to NB cell proliferation. Both siRNA1 and siRNA2 exhibited effective knockdown efficiency, and were confirmed to inhibit cell proliferation; siRNA1 was used for subsequent experiments.

To explore whether SNHG16 affects cell migration, a wound healing assay was conducted using SH-SY5Y cells wounded with a micropipette tip. As shown in Fig. 4A, the wider width in the SNHG16 silencing group suggested that SNHG16 knockdown significantly suppressed the migratory ability of SH-SY5Y cells. Further quantification analysis revealed a significant difference in the width between the SNHG16 silencing group and the control group at 48 and 72 h after wounding (Fig. 4B). Furthermore, the results of a Transwell assay further indicated that the migratory capacity of SH-SY5Y cells transfected with siRNA-SNHG16 was markedly decreased compared with the control group (Fig. 4C). These results suggested that SNHG16 knockdown inhibited migration of SH-SY5Y cells.

The cell cycle is closely associated with cell proliferation; therefore, flow cytometry was used to investigate the potential effects of SNHG16 on cell cycle alterations. The percentage of cells in G₀/G₁ phase was significantly increased, whereas the percentage in S phase was decreased in the SNHG16-knockdown group compared with in the control group (Fig. 5A and B; Table II). These results demonstrated that SNHG16 silencing in SH-SY5Y cells induced cell cycle arrest at the G₀/G₁ phase.

Cellular apoptosis was measured by AO/EB staining following siRNA transfection. As shown in Fig. 6A, the cells in both groups appeared uniformly green with no distinct red-orange staining, indicating no detectable apoptotic cells. However, both cell number and density were reduced in the SNHG16-knockdown group, suggesting that SNHG16 silencing inhibited cell proliferation without apoptosis. Detection of caspase-3/7 activity also revealed that SNHG16 knockdown had no significant effect on caspase-3/7 activity (Fig. 6B). These results indicated that SNHG16 knockdown did not induce apoptosis in NB.

To explore the underlying molecular mechanism of SNHG16 function, starBase v2.0 was used to identify potential RBPs that bind SNHG16 (34). Predicted target proteins of SNHG16 are listed in Table SI, and functional enrichment analysis was conducted by Cytoscape. SNHG16-related RBPs were revealed to be mainly involved in the regulation of mRNA metabolic processes, gene silencing by miRNA, mRNA transport, RNA splicing and translation (Fig. 7A-C), implying that the molecular mechanism underlying the effects of SNHG16 is associated with RNA-related processes.