Human NB samples (n=37; male/female ratio, 17/20; age, 3±0.45 years) and matched adjacent normal tissues were randomly obtained from patients who underwent curative resection at the Department of Spine Surgery, Hong-Hui Hospital (Xi'an, China) between 2012 and 2013. All samples were immediately frozen in liquid nitrogen. None of the patients were pretreated with radiotherapy or chemotherapy prior to surgery. The diagnoses of these tissue samples were all verified by pathologists at Hong-Hui hospital. All samples were obtained with informed consent, and the study protocol was approved by the Ethics Committee of Hong-Hui Hospital (Xi'an Jiaotong University College of Medicine, Xi'an, China).

All clinical specimens were stored at −80°C and homogenized using a homogenizer (Invensys Systems APS, Albertslund, Denmark) at 500 × g at 4°C prior to extraction of RNA using a mirVana™ miRNA Isolation kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) by centrifugation (12,000 × g, 4°C, 15 min), according to the manufacturer's protocol. The RNA concentration and quality were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.), and 1 µg RNA was used for reverse transcription (RT). A RT primer specific for miR-451 (5′-CGCTACGTAACGGCATGACAGTG(T)24-3′; Ambion, Austin, TX, USA) was used to produce cDNA from the miR in the RT procedure. Subsequently, quantitative-polymerase chain reaction (qPCR) was performed using SYBR Green PCR master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR reaction mixture was composed of 1 µl cDNA, 0.5 µl primer and 5 µl SYBR Green PCR master mix buffer (total volume, 10 µl). qPCR was performed over 30 cycles of 30 sec at 98°C, 90 sec at 58°C and 20 sec at 72°C; with a final extension at 72°C for 5 min using a LightCycler^® (Roche Diagnostics GmbH, Mannheim, Germany). The primers were miR-451 forward, 5′-AAACCGTTACCATTACTGAGTT-3′ and reverse, 5′-CGCTACGTAACGGCATGACAGTG-3′. The relative quantification of miR-451 was calculated using the 2^−ΔΔCq method (18). The data were normalised using U6 as an internal control, and were measured relative to a calibrator sample as the external control.

The SK-N-SH and GI-LA-N human NB cell lines were obtained from the American Type Culture Collection and were grown in Eagle's minimum essential medium (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin/streptomycin (Invitrogen), in a cell culture incubator at 37°C and 5% CO₂. The miR-451 mimics and control miR mimics were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The cells (5×10⁴ cells/well) were transfected with the miR mimic (30 nM) or the macrophage migration inhibitory factor (MIF) recombinant plasmid (200 ng) using Lipofectamine™ 2000 reagent (Invitrogen) at 37°C, according to the manu facturer's protocol. All assays were performed 48 h following transfection.

To determine the cell proliferation capacity, the cells were monitored using cell growth curves. The cells were seeded into 96-well microplates (10³ cells/well), and cell viability was monitored using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay (Sigma-Aldrich, St. Louis, MO, USA) at indicated time points (1, 2, 3, 4 and 5 days following seeding into plates). The absorbance value (A) was read at 570 nm using a Benchmark Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The transfected cells (5×10⁴ cells/well) were seeded into 6-well plates for 48 h, collected by centrifugation (500 × g, 4°C, 5 min), washed with cold phosphate-buffered saline (PBS) and fixed with 70% cold ethanol at 4°C overnight. The fixed cells were then collected, washed with PBS and resuspended in fluorescence-Activated Cell Sorting (FACS) solution, containing PBS, 0.1% TritonX-100 (Sigma-Aldrich), 60 µg/ml propidium iodide (PI; Sigma-Aldrich), 0.1 mg/ml DNase-free RNase (Sigma-Aldrich) and 0.1% trisodium citrate (Sigma-Aldrich). After a final incubation on ice for 30 min, cell cycle analysis was performed using a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using CellQuest 5.2.1 (BD Biosciences) or ModFit 3.0 software (Verity Software House, Inc., Topsham, ME, USA) packages.

An Annexin-V fluorescein isothiocyanate (FITC) Apoptosis kit (Cell Signaling Technology, Inc., Danvers, MA, USA) was used to evaluate apoptosis. Briefly, the cells were seeded into six-well culture plates at 5×10⁴ cells/well and transfected for 48 h, following which they were harvested by trypsinization and washed with cold PBS. Immediately following re-suspension of the cells in medium, Annexin-V FITC and PI buffers (KaiGi Technology, Guangzhou, China) were incubated with the cells for 30 min at 4°C in the dark. The Annexin V-FITC and PI signals were detected using flow cytometry (BD FACSCalibur; BD Biosciences).

Transwell migration assays were performed using 6.5-mm diameter cell culture inserts (8-µm pore size; BD Biosciences) in 24-well culture plates. A total of 2×10⁴ suspended cells (per well) were seeded into the upper chamber of the Transwell insert, which was pre-coated with 1 mg/ml Matrigel (BD Biosciences). In the lower part of the chamber, 0.8 ml medium with 20% FBS was added. The inserts were washed with PBS following incubation for 24 and 48 h at 37°C, and the adherent cells on the membrane of the upper chamber were removed from the upper surface using a cotton swab. The cells on the lower chamber membrane were fixed in cold methanol and stained with 2% crystal violet (Sigma-Aldrich).

For the scratch-wound assay, the cells were seeded into 24-well plates (5×10⁵ per well) and cultured until confluent. The cell monolayers were wounded by manually scratching their surface with a 200-µl pipette tip. The cells were then washed several times in culture medium to remove cell debris, and the culture was continued in fresh medium. After 12 h incubation at 37°C, the migration of the cells was evaluated by counting the number of migrated cells under a phase contrast microscope (Eclipse 90i; Nikon, Tokyo, Japan).

The cells were lysed on ice using radio-immunoprecipitation assay buffer, containing 150 mM NaCl, 1% sodium deoxycholate, 1% NP40, 0.1% sodium dodecyl sulfate, 50 mM Tris (pH 8.8), 10 mM NaF, 1X protease inhibitors and phenylmethanesulfonyl fluoride. Protein concentrations were measured using a bicinchoninic acid assay method, and 30 µg of protein was separated by 10% SDS-polyacrylamide gel electrophoresis (Sigma-Aldrich), followed by transfer onto polyvinylidene fluoride membranes (Durapore^®; Thermo Fisher Scientific) for analysis. The membranes were then blocked with 5% skimmed milk at room temperature for 1 h and washed with Tris-buffered saline containing Tween 20 three times for 5 min each. Rabbit anti-human MIF polyclonal antibody was used as the primary antibody (cat. no. ab7207; 1:500 dilution; Abcam, Cambridge, MA, USA). Rabbit anti-human GAPDH monoclonal antibody (cat. no. 5174; 1:1,000 dilution, Cell Signaling Technology, Inc.) was used as a control. The bands were detected using an Enhanced Chemiluminescence kit (GE Healthcare Life Sciences, Chalfont, UK) and visualised using a ChemiDoc XRS system (Bio-Rad Laboratories, Inc.).

To generate reporter constructs for the luciferase assays, the full-length 3′-untranslated region of MIF, as well as the mutant sequence of MIF, were synthesized using PCR. The sequences of the primers used were as follows: MIF 3′UTR, forward 5′-GGGGTACCCCAGAGCCGCAGGGACCCA-3′ and reverse 5′-GAAGATCTTCAAGTCTCTAAACCGTTT-3′; and Mut MIF 3′UTR, forward 5′-TGGTGGGGAGAAATAAGTGCTTTAGAGACT-3′ and reverse 5′-AGTCTCTAAAGCACTTATTTCTCCCCACCA-3′. The PCR-amplified MIF 3′UTR fragment was digested with KpnI and BglII restriction enzymes at 37°C for 6 h and then cloned into the restriction sites downstream of the luciferase open reading frame in the pGL3-promoter Luciferase vector (Invitrogen) at 4°C overnight using ligase.

The 3′-UTR deletion of MIF was amplified using PCR with the following primers: Forward 5′-GCCATCATGCCGATGTTCATCG-3′ and reverse 5′-CGGCTCTTAGGCGAAGGTGGAG-3′. The resulting PCR amplicons of MIF were cloned into the T vector (Promega Corporation, Madison, WI, USA). The clones were confirmed by sequencing.

For the Fluorescent reporter assay, the human NB cells were seeded into a 24-well plate (1.5×10⁵ per well) and co-transfected with the miR mimics (50 nM) and a reporter recombinant plasmid (100 ng) using Lipofectamine™ 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Luciferase activity was analysed 48 h following co-transfection using dual luciferase assays (Promega Corp.), and values were normalised against the activity of the Renilla luciferase gene.

All values are expressed as the mean ± standard deviation from at least three repeated individual experiments for each group. The data obtained in the present study were analyzed using the statistical package SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Significant differences were analysed using Student's t-test or χ² analysis for comparisons between two groups, and one-way analyses of variance or the non-parametric Kruskal-Wallis H test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To investigate the pathogenicity of miR-451 in the development and progression of NB, 37 NB tissue samples were analysed using RT-qPCR. The clinicopathologic data for 37 patients are presented in Table I. Based on the overall expression level of miR-451, the NB specimens were divided into two groups, a high miR-451 expression level group and a low expression level group. The results showed that the expression of miR-451 in the NB tissues was significantly lower, compared with that in the matched paraneoplastic tissues (P=0.0001). Additionally, significant correlations were found between the reduction in miR-451 and unfavourable variables, including carcinoma size (P=0.0081), level of differentiation (P=0.0217), TNM stage (P=0.0220), lymph node metastasis (P=0.0489) and distant metastases (P=0.0201). No significant difference was observed when the groups were divided by the clinicopathologic features of gender and age.

To investigate the role of miR-451 in human NB, miR-451 was overexpressed in SK-N-SH and GI-LA-N human NB cells by transfection with miR-451 mimics. The RT-qPCR results showed that transfection with the miR-451 mimics markedly elevated the levels of miR-451 in the SK-N-SH and GI-LA-N cells (Fig. 1A). The MTT assays showed that the overexpression of miR-451 significantly inhibited the growth of the SK-N-SH and GI-LA-N cells, and this effect increased with time (Fig. 1B). To further elucidate the antigrowth mechanism of miR-451 in NB cells, the present study analysed cell cycle and apoptosis. Overexpression of miR-451 in the NB cells significantly increased the percentage of cells in the G₀/G₁ phase of the cell cycle, compared with the control group (Fig. 1C). However, no significant difference were observed in the rates of apoptosis following transfection with the miR-451 mimics in the SK-N-SH and GI-LA-N cells (Fig. 1D).

The effect of miR-451 on the invasion and migration of human NB cells was evaluated using Transwell and scratch-wound assays. The Transwell assays showed that transfection with the miR-451 mimics reduced the number of invasive cells in the SK-N-SH and GI-LA-N cell lines, compared with the control group (Fig. 2A and B). In addition, the scratch-wound assays also revealed that the migratory abilities of the SK-N-SH and GI-LA-N cells were significantly reduced following transfection with the miR-451 mimics (Fig. 2C and D).

As miRs exert their function predominantly through the inhibition of target genes, the present study detected the target of miR-451 in NB cells. Previous studies have reported that MIF serves as a target of miR-451 post-transcriptional repression in gastrointestinal and nasopharyngeal carcinoma (14,19). To demonstrate whether miR-451 regulated the expression of MIF in NB, the SK-N-SH and GI-LA-N cells were transfected with miR-451 mimics. Western blot analysis confirmed that transfection with the miR-451 mimics significantly reduced the protein expression of MIF in the NB cells (Fig. 3A). To demonstrate that the inverse correlation between miR-451 and the expression of MIF in NB cells was due to a direct interaction, the potential seed sequence for miR-451 in the 3′UTR region of MIF was analysed, and the wild-type and mutant MIF 3′UTR fragments were cloned into a luciferase reporter gene system (Fig. 3B). The MIF 3′-UTR luciferase reporter plasmid and the miR-451 or control mimics were co-transfected into the SK-N-SH and GI-LA-N cells. The results revealed a 38.5 and 46.9% reduction in luciferase activity in the miR-451-transfected SK-N-SH cells and GI-LA-N cells, respectively, compared with the cells transfected with the control, respectively. However, no alteration in luciferase activity was detected with the mutant MIF 3′-UTR luciferase reporter plasmid (Fig. 3C and D).

To examine whether the repression of miR-451 on NB cell growth and invasion was mediated through MIF, the present study co-transfected the NB cells with expression vectors for MIF lacking the respective 3′UTR, or empty vectors, with the miR-451 mimics. Western blot analysis demonstrated increased protein expression of MIF in the NB cells co-transfected with this MIF vector, compared with those transfected with the empty vector (Fig. 4A). The re-expression of MIF led to the apparent rescue of NB cell proliferation, determined from the results of the MTT assay, with no significant differences, compared with the control (Fig. 4B). Cell cycle analysis revealed that the re-expression of MIF decreased the percentage of cells in the G₀/G₁ phase and reversed the growth inhibitory effect of miR-451 (Fig. 4C). Similarly, the Transwell and scratch-wound assays showed that the re-expression of MIF reversed the inhibitory effects of miR-451 on invasion and migration (Fig. 4D and E).