The nephroblastoma cell lines, GHINK-1, WiT49, 17–94 and HFWT, and 293T cells were obtained from the American Type Culture Collection. The cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO₂ incubator.

The association between NUSAP1 and HOXB2 was predicted by bioinformatics analysis using the JASPAR database (jaspar.genereg.net).

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and was then reverse transcribed into cDNA using MMLV reverse transcriptase (Promega Corporation) according to the manufacturer's specifications. qPCR analysis was carried out using SYBR Green mix (Thermo Fisher Scientific, Inc.). The PCR thermocycling conditions applied were as follows: Initial holding period at 95°C for 30 sec, followed by 40 cycles of 94°C for 5 sec and 60°C for 30 sec. GAPDH served as an internal reference gene. The primer sequences used were as follows: NUSAP1 forward, 5′-CGTCCCCTCAACTATGAACCAC-3′ and reverse, 5′-GCGTTTCTTCCGTTGCTCTT-3′; HOXB2 forward, 5′-CGCCAGGATTCACCTTTCCTT-3′ and reverse, 5′-CCCTGTAGGCTAGGGGAGAG-3′; and GAPDH forward, 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse, 5′-AGGGGCCATCCACAGTCTTC-3′. The quantification was performed using the 2^−ΔΔCq method (16).

Cells were lysed with RIPA buffer (Beyotime Institute of Biotechnology) with phosphatase inhibitor AEBSF (Beyotime Institute of Biotechnology) on ice for 30 mins. Following centrifugation at 350 × g for 20 min at 4°C, a BCA protein assay kit (Sigma-Aldrich; Merck KGaA) was utilized to quantify the protein concentration. Proteins (40 µg/lane) were separated by 10% SDS-PAGE and were then transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.). Following blocking with 5% skimmed milk for 1.5 h at room temperature, the membranes were incubated with primary antibodies against NUSAP1 (1:1,000; cat. no. ab137230), HOXB2 (1:1,000; cat. no. ab220390), phosphorylated (p)-PI3K (1:1,000; cat. no. ab191606), p-Akt (1:1,000; cat. no. ab38449), E-cadherin (1:1,000; cat. no. ab40772), N-cadherin (1:1,000; cat. no. ab76011), Vimentin (1:1,000; cat. no. ab92547), PI3K (1:1,000; cat. no. ab32089), Akt (1:1,000; cat. no. ab18785) and GAPDH (1:1,000; cat. no. ab8245; all from Abcam) at 4°C overnight. The next day, the membranes were washed and incubated with the corresponding secondary antibodies (1:5,000; cat. no. ab150077; Abcam) at room temperature for 1 h. For p-PI3K and p-Akt, after the detection of PI3K and Akt, the same membrane was washed with stripping buffer (cat. no. P0025N; Beyotime Institute of Biotechnology), and the aforementioned protein detection steps were repeated to detect the expression of p-PI3K and p-Akt. Protein signals were detected using ECL detection kit (Amersham; Cytiva) and semi-quantitative analysis was conducted using ImageJ software (version 1.8.0; National Institutes of Health).

HFWT cells were cultured in a 6-well plate at a density of 1×10⁶ cells/well at 37°C in a 5% CO₂ incubator for 24 h. The lentiviral vectors packaging short hairpin RNA (shRNA) NUSAP1 (sh-NUSAP1), negative control shRNA (sh-NC), HOXB2 overexpression plasmid (Ov-HOXB2) and Ov-NC were obtained from Shanghai GeneChem Co., Ltd., and were then transduced into cells at 20 nM using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The transfection efficiency was verified by RT-qPCR and western blot analyses after 48 h.

Following culture for 48 h at 37°C in a 5% CO₂ incubator, HFWT cells in each well at a density of 8×10³ cells/well were supplemented with 20 µl MTT solution and incubated for an additional 3 h. Subsequently, the supernatant was removed and 200 µl DMSO was added into each well to dissolve formazan crystals. The absorbance at 490 nm was detected using a microplate reader (Multiskan Sky; Thermo Fisher Scientific, Inc.).

Cell proliferation was assessed using the Cell-Light EdU DNA Cell Proliferation kit (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's protocol. Briefly, following transfection, the HFWT cells were stained with fluorescent-labeled EdU for 2 h at room temperature. Subsequently, cells were fixed in 4% ethanol for 30 min at room temperature and were then stained with Apollo reaction buffer liquid at 37°C for 30 min in the dark. Image acquisition was performed using a laser confocal microscope (magnification, ×200).

Transfected HFWT cells were cultured normally in a 6-well plate at a density of 1×10³ cells/well. Following incubation for 2 weeks, cells were fixed with methanol for 15 min at room temperature and stained with 0.1% crystal violet for 20 mins at room temperature. Finally, the visible colonies with a diameter >0.5 mm were counted using ImageJ software (version 1.8.0; National Institutes of Health) and images were captured under a microscope.

For wound healing assays, HFWT cells were grown to 100% confluence in 6-well plates after transfection. Subsequently, two linear wounds were created in each well by scratching the monolayer with a sterile pipette tip. The wells were then washed with PBS and cells were cultured in serum-free DMEM for 24 h. To assess cell migration, images of the same areas in the wells were captured at 0 and 24 h using a light microscope following wounding. Wound healing was evaluated by measuring the total surface area covered by the cells.

To assess cell invasion, 24-well Transwell plates (Corning, Inc.) with 8-µm pore inserts were coated with Matrigel (BD Biosciences) at 37°C for 30 min. The transfected HFWT cells (5×10⁵ cells/well) in serum-free DMEM were seeded onto the Matrigel-coated upper chamber, while the lower chamber was supplemented with DMEM containing 10% FBS. Following incubation for 24 h at 37°C, the cells on the upper chamber were gently removed with cotton swabs. The invading cells in the lower chamber were fixed in alcohol for 15 min and stained with 0.1% crystal violet solution for 10 min at room temperature. Finally, the cells were observed under an optical microscope (Olympus Corporation; magnification, ×100).

To generate the luciferase construct, 0.5 µg vectors containing the 3′-untranslated region (UTR) of wild-type (WT) NUSAP1 or mutant (MUT) 3′-UTR NUSAP1using a QuickMutation kit (Beyotime Institute of Biotechnology), with control vector or HOXB2 overexpression vector and pMIR-Renilla vector (Shanghai GeneChem Co., Ltd) were synthesized and cloned into GV272 vectors by GeneChem (China). HFWT cells were co-transfected using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) with WT or MUT 3′-UTR of NUSAP1 (NUSAP1-WT or NUSAP1-MUT, respectively) and Ov-HOXB2 or Ov-NC for 48 h. A site directed mutagenesis kit was used to mutant 3′-untranslated region of NUSAP1. Following transfection, the relative luciferase activity was determined after 48h using the Dual-Luciferase Reporter Assay kit (Promega Corporation), according to the manufacturer's instructions. Renilla luciferase activity was used to normalize the firefly luciferase activity.

The ChIP assay was carried out using the Chromatin Immunoprecipitation Assay Kit (MilliporeSigma), according to the manufacturer's instructions. Briefly, the cells were lysed with 100 µl SDS lysis buffer (Beyotime Institute of Biotechnology) and 500 µl of ChIP sonication nuclear lysis buffer was added to cell particles and centrifuged at 1,000 × g at 4°C for 5 mins. Then, 10 µg fragmented chromatin was incubated with 2 µg anti-IgG (cat. no. A-11031) and 5 µg anti-HOXB2 (cat. no. PA5-101640) antibodies (1:500; both Thermo Fisher Scientific, Inc.). Subsequently, 20 µl protein A+G agarose beads (Beyotime Institute of Biotechnology) was added and incubated for another 4 h at 4°C. After the supernatant was removed, the collected agarose beads were washed five times with 100 µl PBS (0.01 M; pH 7.4) and denatured by boiling for 5 min. Total RNA was then subjected to reverse transcription to cDNA using SMART MMLC Reverse Transcriptase (Takara Biotechnology Co., Ltd.). The recruited DNA was then subjected to PCR using primers detecting the distal enhancer regions of NUSAP1 using an ABI 7500 system (Thermo Fisher Scientific, Inc.). Subsequently, the PCR products were electrophoresed in 1% agarose gel, stained in ethidium bromide for ~15 min, rinsed with water and photographed by gel imaging system. Gel images and positive bands with average optical density values were analyzed by Gel optical density analysis software Gel pro4.0. The resulting data were normalized to housekeeping gene β-actin.

All data are expressed as the mean ± SD of three independent experimental repeats. All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, Inc.). The differences between multiple groups were compared by one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The mRNA and protein expression levels of NUSAP1 in nephroblastoma cells were detected via RT-qPCR and western blot analyses, respectively. The results showed that the expression of NUSAP1 was significantly increased in nephroblastoma cell lines compared with 293T cells (Fig. 1A and B). The expression of NUSAP1 was more notably increased in HFWT cells. Therefore, HFWT cells were used for the subsequent experiments. Subsequently, the expression of NUSAP1 was knocked down in HFWT cells following cell transfection with sh-NUSAP1 and the transfection efficiency was evaluated using RT-qPCR and western blot analysis. As shown in Fig. 1C and D, the expression of NUSAP4 was significantly decreased in cells transfected with shRNA-NUSAP1#2 and this shRNA clone was therefore used for the subsequent experiments. To evaluate cell viability, cells were first divided into the control, sh-NC and sh-NUSAP1 groups followed by MTT assay. The results showed that cell viability was significantly reduced in the sh-NUSAP1 group compared with the sh-NC group (Fig. 1E). Cell proliferation was assessed using colony formation assay. As shown in Fig. 1F, compared with the sh-NC group, the proliferative ability of cells was significantly decreased in the sh-NAUSAP1 group. The results of EdU staining were consistent with those obtained with the colony formation assay (Fig. 1G). The aforementioned findings suggested that NUSAP1 silencing could attenuate the proliferation of nephroblastoma cells.

Subsequently, wound healing and Transwell assays were carried out to evaluate the migratory and invasive abilities of nephroblastoma cells, respectively. The results demonstrated that the migratory and invasive abilities of nephroblastoma cells were notably decreased in the sh-NAUSAP1 group compared with the sh-NC group (Fig. 2A and B). Subsequently, western blot analysis was performed to detect the expression levels of epithelial-to-mesenchymal transition (EMT)-related proteins. The analysis showed that, compared with cells transfected with sh-NC, the expression of E-cadherin was significantly increased, while that of N-cadherin and vimentin was notably reduced in cells in the sh-NUSAP1 group (Fig. 2C). Therefore, it was hypothesized that NUSAP1 silencing could inhibit the metastasis of nephroblastoma cells.

Bioinformatics analysis using the JASPAR database predicted a HOXB2 binding site on the NUSAP1 gene promoter (Fig. 3A and B). Subsequently, the expression of HOXB2 in nephroblastoma cells was assessed using RT-qPCR and western blot analyses. It was observed that HOXB2 was significantly upregulated in HFWT cells compared with 293T cells (Fig. 3C and D). Then, transfection experiments were performed to overexpress HOXB2 and the transfection efficiency was also determined. HOXB2 was significantly upregulated in the Ov-HOXB2 group compared with the Ov-NC group (Fig. 3E and F). The interaction between HOXB2 and NUSAP1 was verified by dual-luciferase reporter assay (Fig. 3G), and was further confirmed using a ChIP assay (Fig. 3H). In addition, the expression of NUSAP1 in the Ov-HOXB2 group was significantly increased after overexpression of HOXB2 compared with the Ov-NC group (Fig. 3I). Subsequently, cells were divided into the control, sh-NC, sh-NUSAP1, sh-NUSAP1 + Ov-NC and sh-NUSAP1 + Ov-HOXB2 groups and the expression of NUSAP1 was then determined. The results showed that HOXB2 overexpression could reverse the sh-NUSAP1-mediated reduced NUSAP1 expression (Fig. 3J and K). These findings indicated that HOXB2 could promote the expression of NUSAP1.

MTT, EdU and colony formation assays were performed to evaluate cell proliferation. Therefore, compared with the sh-NUSAP1 + Ov-NC group, the proliferative ability of nephroblastoma cells in the sh-NUSAP1 + Ov-HOXB2 group was significantly enhanced (Fig. 4A-D). Additionally, wound healing and Transwell assays showed that compared with the sh-NUSAP1 + Ov-NC group, the invasive and migratory abilities of nephroblastoma cells in the sh-NUSAP1 + Ov-HOXB2 group were also significantly increased (Fig. 5A and B). To assess the expression levels of EMT-related proteins, western blot analysis was carried out. The analysis showed that compared with the sh-NUSAP1 + Ov-NC group, the expression of E-cadherin was decreased, while that of N-cadherin and Vimentin was increased in the sh-NUSAP1 + Ov-HOXB2 group (Fig. 5C). These findings suggested that HOXB2 overexpression could reverse the inhibitory effect of NUSAP1 knockdown on the proliferation and metastasis of nephroblastoma cells.

Western blot analysis revealed that the expression levels of the PI3K/Akt pathway-related proteins were abnormally altered. Therefore, the expression levels of p-PI3K and p-Akt were significantly decreased in the sh-NUSAP1 group compared with the shRNA-NC group. By contrast, p-PI3K and p-Akt were significantly upregulated in the sh-NUSAP1 + Ov-HOXB2 group compared with the sh-NUSAP1 + Ov-NC group (Fig. 6). The aforementioned findings suggested that HOXB2 overexpression could reverse the inhibitory effect of NUSAP1 silencing on the proliferation and metastasis of nephroblastoma cells, possibly via the PI3K/Akt signaling pathway.