The human neuroblastoma SH-SY5Y cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). SH-SY5Y cells were cultured at 37°C in a 5% CO₂ atmosphere in Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal bovine serum (FBS), 100 U/ml streptomycin and 100 U/ml penicillin (all Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The colorimetric water-soluble tetrazolium salt assay was applied to detect cell proliferation using a Cell Counting kit-8 (CCK8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan), according to the manufacturer's protocol. SH-SY5Y cells were seeded into 96-well plates at a density of 1×10³ cells/well, and cell proliferation was evaluated at 12, 24, 48, 72 and 96 h. The number of viable cells was assessed by measuring the absorbance at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

SH-SY5Y cells were maintained in complete minimum essential medium supplemented with 10% FBS and 1% penicillin/streptomycin (all Gibco; Thermo Fisher Scientific, Inc.) and the cells were plated in 6-well plates at a density of 1,000 cells/well followed by incubation at 37°C overnight. The media was replaced every 2 days, and after 2 weeks each well was washed with 1 ml PBS followed by adding 1 ml of crystal violet (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) solution (1% crystal violet and 10% ethanol) into each well. After 10 min incubation at room temperature, the colonies were washed out with PBS three times and were counted by eye.

Cell cycle analysis of SH-SY5Y cells was performed using the BD Cycle assay kit (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol. Different groups of SH-SY5Y cells were seeded into flasks at a density of 7×10⁴ cells (T25 flasks; Thermo Fisher Scientific, Inc.) and incubated at 37°C for 48 h. Following incubation, the cells were harvested and fixed at 4°C in 90% methanol for ~30 min. The cells were centrifuged at 4°C for 5 min at 1,000 × g and washed twice with PBS. The pellets were then resuspended in propidium iodide and incubated at 37°C for 1 h. A fluorescence-activated cell sorting machine (FACS Calibur flow cytometer; BD Biosciences) was used and data were analyzed using BD CellQuest Pro software version 4.0.2 (BD Biosciences).

The culture inserts (ibidi GmbH, Munich, Germany), consisting of 2 reservoirs separated by a 500-µm-thick wall, were placed in a 24-well plate. According to the manufacturer's protocol, an equal amount (70 µl) of SH-SY5Y cell suspension (1×10⁶ cells/ml) was added to each reservoir followed by incubation at 37°C. Following complete attachment of the cells (10 h), the culture inserts were gently removed and the medium was replaced with serum-free DMEM-F12 medium containing 0.2% bovine serum albumin (BSA; Beyotime Institute of Biotechnology, Haimen, China). The gap between the two cell layers was observed at 0 and 20 h after Zip8 knockdown under an inverted microscope (Advanced Microscopy Group, Mill Creek, WA, USA).

RT-qPCR was performed to detect the mRNA expression levels of Zip8, glycogen synthase kinase-3 β (Gsk-3β), β-catenin, NF-κ-B essential modulator (Ikbkg), TANK, MMP2, MMP3, MMP9 and MMP14, according to the manufacturer's protocol. Total cellular RNA was extracted from SH-SY5Y cells by lysing cells with TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) reagent followed by centrifugation at 12,000 × g for 15 min at 4°C with chloroform (5:1 ratio). The supernatant was centrifuged with isopropanol (1:1 ratio) at 10,000 × g at 4°C for 10 min. The RNA pellet was washed with 75% ethanol and solubilized with DNase and RNase free water. The RNA was quantified by measuring absorbance at 260 nm using a NanoDrop ND-1000 (Thermo Fisher Scientific, Inc.). Single-stranded cDNA was prepared using a Reverse Transcription system (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. Briefly, 1 µg total RNA was added into the mixture and incubated at 37°C for 15 min, and then kept at 85°C for 5 sec. The mRNA expression of the target gene was determined by SYBR-Green assays. The SYBR-Green qPCR kit was purchased from Roche Diagnostics GmbH (Mannheim, Germany). qPCR was performed using an Applied Biosystems 7300 sequence detection system. The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 45 cycles at 95°C for 15 sec and at 60°C for 25 sec. All experiments were performed in triplicate. The relative gene expression levels were calculated using the 2^−ΔΔCq method (21) and normalized to GAPDH. The following primers were used: Zip8 forward, 5′-ATGCTACCCAAATAACCAGCTC-3′ and reverse, 5′-ACAGGAATCCATATCCCCAAACT-3′; Gsk3β forward, 5′-AGACGCTCCCTGTGATTTATGT-3′ and reverse, 5′-CCGATGGCAGATTCCAAAGG-3′; β-catenin forward, 5′-CATCTACACAGTTTGATGCTGCT-3′ and reverse, 5′-GCAGTTTTGTCAGTTCAGGGA-3′; Ikbkg forward, 5′-CGGCAGAGCAACCAGATTCT-3′ and reverse, 5′-CCTGGCATTCCTTAGTGGCAG-3′; TANK forward, 5′-AGCAGAGAATACGTGAACAACAG-3′ and reverse, 5′-CAGAAGCAATGTCTACCTTTGGT-3′; MMP2 forward, 5′-CTGCGGTTTTCTCGAATCCATG-3′ and reverse, 5′-GTCCTTACCGTCAAAGGGGTATCC-3′; MMP3 forward, 5′-CTGGACTCCGACACTCTGGA-3′ and reverse, 5′-CAGGAAAGGTTCTGAAGTGACC-3′; MMP9 forward, 5′-GAGGCGCTCATGTACCCTATGTAC-3′ and reverse, 5′-GTTCAGGGCGAGGACCATAGAG-3′; MMP14 forward, 5′-CTTCCGTGGAAACAAGTACTACCGT-3′ and reverse, 5′-ATCCCTTCCCAGACTTTGATGTTC-3′; and GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′.

The protein expression levels of ZIP8, NF-κB, IκB, phosphorylated (p)-IκB, and MMP2, 3, 9 and 13, histone H1 and GAPDH in SH-SY5Y cells were evaluated by western blot analysis. Total proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The cells were washed twice with ice-cold PBS and lysed using a radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Total cell lysates were then centrifuged at 12,000 × g for 15 min at 4°C and the supernatants were used for further processing. The bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology) was used to determine the protein concentration. Total proteins (20 µg) were separated by 10% SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% BSA for 2 h at room temperature and incubated overnight at 4°C with different primary antibodies: Anti-Zip8 (cat. no. ab106576; dilution 1:1,000), anti-NF-κB (cat. no. ab32360; dilution 1:1,000), anti-IκB (cat. no. ab32518; dilution 1:1,000), anti-p-IκB (cat. no. ab12135; dilution 1:1,500), anti-MMP2 (cat. no. 37150; dilution 1:1,000), anti-MMP3 (cat. no. 52915; dilution 1:2,000), anti-MMP9 (cat. no. 38898; dilution 1:1,000), anti-MMP14 (cat. no. 51074; dilution 1:5,000), anti-histone H1 (cat. no. 61177; dilution 1:1,000) and anti-GAPDH antibody (cat. no. ab181602; dilution 1:10,000; all Abcam, Cambridge, UK). Following incubation with primary antibodies, membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (cat. no. 7074; dilution 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescent assay kit (Beyotime Institute of Biotechnology) and a luminescent image analyzer.

The short hairpin (sh)RNA sequences that were used were as follows: Zip8-shRNA1 forward, 5′-GCCAAGTTCATGTACCTGTTTCTCGAGAAACAGGTACATGAACTTGGCTTTTT-3′ and reverse, 5′-AAAAAGCCAAGTTCATGTACCTGTTTCTCGAGAAACAGGTACATGAACTTGGC-3′; Zip8-shRNA2 forward, 5′-CCTTGCTATTCAACTTCCTTTCTCGAGAAAGGAAGTTGAATAGCAAGGTTTTT-3′ and reverse, 5′-AAAAACCTTGCTATTCAACTTCCTTTCTCGAGAAAGGAAGTTGAATAGCAAGG-3′; scramble control shRNA forward, 5′-CTCTTCTACTCATCCACTTTTCTCGAGAAAAGTGGATGAGTAGAAGAGTTTTT-3′ and reverse, 5′-AAAAACTCTTCTACTCATCCACTTTTCTCGAGAAAAGTGGATGAGTAGAAGAG-3′. The sequences were synthesized (Shanghai GeneChem Co., Ltd., Shanghai, China) and inserted into the lentiviral vector PLVX-IRES-ZsGreen1 by Shanghai GeneChem Co., Ltd., through standard molecular cloning methods and the recombinant plasmids were confirmed by sequencing. The lentiviral plasmids including PLVX-IRES-ZsGreen1 vector, pVSVG, pREV and pRRE were purchased from Takara Biotechnology Co., Ltd. HEK293T cells (Shanghai GeneChem Co., Ltd.) were cultured in 10-cm dishes at a concentration of 6×10⁵ cells/ml for 24 h at 37°C and 5% CO₂ in a humidified incubator. Recombinant lentiviral particles were then generated by co-transfecting the packaging plasmids (5 µg for each plasmids), including packaging plasmids (pVSVG, pREV and pRRE), and the shRNA1-, shRNA2- or scramble shRNA-containing vector plasmids, into HEK293T cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as the transfection reagent. Following incubation for 48 h at 37°C and 5% CO₂ in a humidified incubator, the lentiviral particles were harvested and centrifuged at 50,000 × g for 2 h at 4°C. SH-SY5Y cells were divided into 3 groups: Scramble control shRNA, Zip8 shRNA1 and Zip8 shRNA2. For the lentiviral infection, the SH-SY5Y cells were seeded onto 6-well plates at a density of 2×10⁴ cells/well. A total of 1 ml of medium containing polybrene (5 µg/ml) and 100 µl lentivirus (virus titer, 10⁹ TU/ml) were added into each well. After 24 h, the medium was replaced with fresh medium (DMEM-F12 supplemented with 10% fetal bovine serum, 100 U/ml streptomycin and 100 U/ml penicillin; Gibco; Thermo Fisher Scientific, Inc.) and incubated for another 48 h. The cells were then examined under an inverted fluorescence microscope (IX71; Olympus Corporation, Tokyo, Japan) and the green fluorescence emitted by the green fluorescent protein in successfully infected cells was observed.

All the results represented at least three independent experiments and the data are expressed as the mean ± standard deviation. Differences between the control and treatment groups were analyzed using one-way analysis of variance followed by Fisher's least significant difference test and SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the function of Zip8 in neuroblastoma, we knockdown the Zip8 expression in SH-SY5Y cells using lentiviral-mediated RNA interference (RNAi). Zip8 shRNA targets were cloned into lentivirus vectors and the lentivirus was further packaged to infect SH-SY5Y cells. The infecting efficiency was more than 90% as assessed by GFP florescence suggestion of the successful lentivirus infection in neuroblastoma cancer cells (Fig. 1). Quantification analysis by RT-qPCR assay revealed that lentivirus-mediated RNAi obviously reduced the Zip8 mRNA expression levels in SH-SY5Y cells (Fig. 2A). The protein levels of Zip8 were also decreased significantly in SH-SY5Y cells infected by Zip8 shRNA lentivirus (Fig. 2B). Hence, the results demonstrated that the expression of Zip8 was effectively silenced by Zip8 shRNA lentivirus in neuroblastoma cell lines.

The effect of Zip8 on neuroblastoma cell proliferation was assessed by CCK-8 assay. According to the results, the proliferation rates of Zip8 silenced SH-SY5Y cells were both obviously reduced compared with that of the scramble control group (P<0.001; Fig. 2C). In addition, the colony formation assay was also performed to evaluate the effect of Zip8 knockdown on the colony formation ability of SH-SY5Y cells. Compared to the scramble control group, the number of cell colonies by crystal violet staining in the Zip8 silenced groups was obviously reduced (Fig. 2D and E). Therefore, these results indicated that Zip8 played an important role in the regulation of neuroblastoma cells proliferation.

The observation that Zip8 knockdown inhibited the proliferation of neuroblastoma cells led to the investigation of whether Zip8 participates in regulation of the cell cycle. The cell cycle distribution was analyzed by flow cytometry following silencing of Zip8 (Fig. 3A). In the G0/G1 phase of cell cycle, the percentages of SH-SY5Y cells accumulated following Zip8 knockdown by lentiviral-mediated RNAi and the scramble control group presented no difference (Fig. 3B). Similarly, there was no significantly difference between each groups in the S phase (Fig. 3C). In the G2/M phase of cell cycle the percentages of SH-SY5Y cells following silencing Zip8 were increased, suggesting G2/M phase arrest following Zip8 depletion (Fig. 3D). Therefore, the results revealed that knockdown of Zip8 suppressed the growth of neuroblastoma cells possibly through induction of cell cycle arrest.

To reveal the essential molecular mechanism involved in the inhibition of neuroblastoma cells proliferation induced by Zip8 silencing, a number of known key genes which serve key roles in regulating cancer cells proliferation, including Gsk-3β, β-catenin, Ikbkg and TANK, were evaluated through RT-qPCR assays in SH-SY5Y cells. As the results demonstrated, the alterations in the mRNA levels of Gsk-3β and β-catenin in each group were not significant (Fig. 4A and B). The mRNA levels of Ikbkg and TANK involved in the NF-κB signaling pathway decreased markedly following Zip8 silencing (Fig. 4C and D). Therefore, western blotting was subsequently performed to evaluate the effect of Zip8 silencing on the NF-κB signaling pathway in each group. The protein levels of nuclear NF-κB were reduced following Zip8 knockdown (Fig. 4E). The NF-κB and IκB levels of Zip8-silenced groups in the cytoplasm were increased; however, p-IκB levels were decreased in cytoplasm (Fig 4F). These results suggested that silencing of Zip8 had an essential effect on the role of the NF-κB signaling pathway in neuroblastoma cell proliferation.

To examine whether Zip8 serves a role in regulating the migration of SH-SY5Y cells, a cell migration assay was performed. The gap between 2 cell layers was observed and recorded at 0 h and 20 h, respectively. The control group of SH-SY5Y cells migrated towards to the empty area after culturing for 20 h. However, the cells of the Zip8-silenced groups demonstrated a marked reduction in their migratory ability, as the gaps between the two cell layers were obviously increased compared with that of the control group after 20 h, suggesting that Zip8 knockdown reduced cell migration (Fig. 5). These data indicated that Zip8 could affect the migration of neuroblastoma cells.

Based on the above data demonstrating that knockdown of Zip8 inhibited the migratory ability of neuroblastoma cells, the key molecular mechanism involved in the phenomenon was investigated. The gene expression levels of migratory-associated proteins, including MMP2, 3, 9 and 14, were detected by RT-qPCR. The results suggested that, the expression levels of all the four MMPs decreased markedly (Fig. 6A-D). The western blot analysis further reconfirmed that the expression levels of MMP2, 3, 9 and 14 were reduced following Zip8 knockdown in SH-SY5Y cells (Fig. 6E). The results demonstrated that Zip8 could regulate the expression of MMPs to affect the migratory ability of neuroblastoma cells.