SHEP1, SK-N-DZ, SK-N-AS, and SK-N-SH human neuroblastoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). SK-N-BE (2), IMR32, BE (2)-C and SY5Y human neuroblastoma cell lines were grown in a 1:1 mixture of DMEM and Ham’s nutrient mixture F12 (F12/DMEM), supplemented with 10% FBS and nonessential amino acids. LAN-6 and SMS-KCNR human neuroblastoma cell lines were grown in RPMI-1640 supplemented with 10% FBS. The growth media and FBS were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). All cells were obtained from American Type Culture Collection (Manassus, VA, USA) and cultured at 37°C in a 5% CO₂ humidified incubator. The 293GPG retroviral packaging cell line was cultured as described previously (28).

The retroviral constructs, pBabe-green fluorescent protein (GFP) and pBabe-GATA3, were used in the overexpression experiments. Retroviruses were produced using the 293GPG packaging cell line as described previously (28). At 24 h following the final round of retroviral infection, cells were cultured in the growth medium containing 1.0 μg/ml puromycin for three days, and drug-resistant cells were pooled. The percentage of retrovirus-infected cells ranged between 80 and 90%, as estimated in parallel infections using the retrovirus-expressing GFP. Over-expression of relevant proteins was verified by an immunoblotting assay.

Following RA (Sigma-Aldrich, St. Louis, MO, USA) treatment or retroviral infection, cells in the exponential growth phase at 70–80% confluence were harvested at various time points and washed once with ice-cold phosphate-buffered saline. Cell pellets were suspended in SDS sample buffer and boiled for 10 min prior to centrifugation at 211 × g for 10 min. Samples were subjected to 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was probed with antibodies and binding was visualized using enhanced chemiluminescence (ECL; Beyotime Institute of Biotechnology, Haimen, China). The following primary antibodies were used: Rabbit polyclonal anti-GATA3 (1:100; H-48, sc-9009; Santa Cruz Biotechnology Inc., Dallas, TX, USA), mouse monoclonal anti-Mash1 (1:100; clone 24B72D11.1; BD Pharmingen, San Diego, CA, USA), rabbit polyclonal anti-peripherin (1:2,000; AB1530; Chemicon International, Inc., Billerica, MA, USA) and mouse monoclonal anti-α-tubulin (1:10,000; B-5-1-2; Sigma-Aldrich). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG (1:5,000, ICN, Bryan, OH, USA) were used as secondary antibodies.

For differentiation assays, RA was dissolved in dimethyl sulfoxide (DMSO) and 10 mM stock solutions were prepared. SK-N-SH cells were treated with 1 μM RA. DMSO (0.1%; Sigma-Aldrich) was used as negative control. Cell growth was observed under a microscope (Olympus IX71; Olympus, Tokyo, Japan) and determined by MTT analysis (Sigma-Aldrich)

Patient data and gene expression datasets were obtained from the Oncogenomics Section Data Center (http://pob.abcc.ncifcrf.gov/cgi-bin/JK). Kaplan-Meier analysis and resulting survival curves were created using GraphPad Prism (version 6.0; GraphPad Software, Inc, La Jolla, CA, USA). All data and P-values (log-rank test) for these experiments were downloaded online (http://pob.abcc.ncifcrf.gov/cgi-bin/JK) and all cutoff values for separating the groups with high and low expression were determined using the online database algorithm (29). A good prognosis of neuroblastoma patients was considered to be associated with better survival.

Cells were mixed in 0.3% Noble agar (Sigma-Alrdich) in DMEM supplemented with 10% FBS and plated at 4,000 cells/well into 6-well plates, which contained a solidified bottom layer composed of 0.6% Noble agar in the same growth medium. At 14 days, colonies were stained with 5 mg/ml MTT and photographed (Olympus, IX71; Olympus). For sphere formation assays, cells were plated at 4,000 cells/well in serum-free DMEM, and supplemented with 20 ng/ml epidermal growth factor and basic fibroblast growth factor (Invitrogen Life Technologies) in Matrigel ultra-low attachment plates (Thermo Fisher Scientific, Pitsburgh, PA, USA). Spheres that arose within 1–2 weeks were counted.

For the tumorigenic assays, six female NOD/SCID mice (4 weeks old) were used and were maintained under SPF conditions. For the tumorigenic assays, the mice were randomly divided into two groups, control group and GATA3-overexpressing group. Mice were injected subcutaneously in both flanks with 1×10⁷ SK-N-SH cells or SK-N-SH-GATA3 cells in 200 μl DMEM. At one week following the injection of tumor cells, tumor growth was estimated using calipers, and tumor volume was calculated using the formula 4/3πr³, where r is the radius of the tumor. Tumors were removed and weighed following three weeks of tumor growth. The present study was approved by the Institutional Animal Care and Use Committee of Southwest University (Chonqing, China).

Data are presented as the mean ± standard deviation. Two-tailed Student’s t-test was conducted for paired samples and was performed using GraphPad Prism version 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P≤0.05 was considered to indicate a statistically significant difference.

The correlation between GATA3 expression levels and prognosis in primary neuroblastoma was investigated using the Seeger microarray dataset, which is available from the online Oncogenomics database. This dataset includes a cohort of 102 neuroblastoma patients with metastatic tumors lacking MYCN amplification (30). Kaplan-Meier analysis of progression-free survival for the Seeger dataset showed that low GATA3 expression was associated with a good prognosis, whereas high GATA3 expression was associated with a poor outcome (Fig. 1A). Furthermore, a box plot of GATA3 expression levels in tumors from patients with either a good or a poor prognosis demonstrated the same result (Fig. 1B). This analysis indicated that GATA3 is a prognostic marker in neuroblastoma, which is independent of the status of MYCN amplification.

The expression of GATA3 in various neuroblastoma cell lines was examined. GATA3 was found to be widely expressed in the majority of neuroblastoma cell lines (Fig. 2A), including BE (2)-C, IMR32, SK-N-DZ, SK-N-AS, and SK-N-BE, which are malignant cell lines. The expression of GATA3 was also investigated in the SHEP1 cell line, which is a benign neuroblastoma cell line with a highly differentiated status (31,32). The results indicated that GATA3 may be associated with the degree of neuroblastoma differentiation and the consequent prognosis. The SK-N-SH cells were a group of mixed cells which were isolated into SY5Y and SHEP1 in different conditions (33), and SY5Y is a type of malignant cell comparing with SHEP1 (32). As hypothesized, GATA3 expression was relatively high in the SY5Y and SK-N-SH cell lines, but there was no detectable expression in the SHEP1 cell line (Fig. 2B). This suggests that GATA3 may be used as a prognostic marker in neuroblastoma.

Since RA is commonly used to induce neuronal differentiation in neuroblastoma (34), SK-N-SH cells were treated with RA for 10 days. On examination the cells displayed morphological features of neuronal differentiation, such as small and rounded cell bodies, scant cytoplasm, and extensive neurite-like processes (Fig. 3A). Furthermore, MTT, sphere formation and soft agar analyses showed that RA treatment resulted in the suppression of cell proliferation, tumorigenicity and self-renewal of neuroblastoma cells (Fig. 3B–E). The GATA3 expression in this process was measured, and the results showed that RA induction led to marked downregulation of GATA3 expression with time (Fig. 3F). These findings suggest that RA-induced neuronal differentiation is accompanied by GATA3 downregulation, which leads to weakened self-renewal of neuroblastoma cells.

To confirm the correlation between GATA3 and self-renewal of neuroblastoma cells, GATA3 was overexpressed in neuroblastoma cells, using GFP as a control (Fig. 4A). GATA3 significantly increased cell proliferation, which was verified by MTT analysis (Fig. 4B). In addition, GATA3 markedly upregulated the self-renewal ability, including the colony forming and sphere forming capability, of neuroblastoma cells (Fig. 4C and 4F). These results demonstrated that high expression of GATA3 is associated with increased self-renewal and cell proliferation in neuroblastoma cells.

Furthermore, the RA-induced neuroblastoma differentiation was accompanied by GATA3 downregulation and the upregulation of peripherin, a neuronal differentiation marker (35) (Fig. 5A). GATA3 upregulation led to a significant increase in the expression of Mash1 (Fig. 5B), which is a potential stem cell or progenitor cell marker (36,37). Similar results were obtained from the in vivo tumorigenicity analysis using the SK-N-SH neuroblastoma cells. Overexpression of GATA3 in SK-N-SH cells significantly enhanced tumor growth and development in NOD/SCID mice (Fig. 5C–E). These data indicate that GATA3 is not only a prognostic marker, but also an important mediator of cell proliferation and differentiation.