A total of 30 samples of nephroblastoma and normal adjacent tissues were obtained from children with nephroblastoma who had undergone surgery at the Department of Pediatric Surgery, Zaozhuang City Hospital between December 2015 and December 2017. All patients did not received chemotherapy or radiotherapy prior to surgery. The present study was approved by the Ethical Committee of the Zaozhuang City Hospital and informed consent was obtained from each child and their guardian.

Nephroblastoma cell line WiT49 was provided by The Hospital for Sick Children (Toronto, Canada) and was subsequently grown in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin streptomycin. These cells were cultured in a humidified chamber at 37°C with 5% CO₂. miR-130b-3p inhibitor (miR-130b-3p antagomir; sequence: 5′-UGCCAACCUUGCAAGCCGAAG-3′) and inhibitor control (antagomir-negative control; sequence: 5′-CAGUACUUUUGUGUAGUACAA-3′) were purchased from GenePharma Co. Ltd. WiT49 cells were transfected with either 100 nM inhibitor control, 100 nM miR-130b-3p inhibitor, 2 µl control-siRNA (cat. no. Sc-36869; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), 2 µl PTEN-siRNA (cat. no. Sc-29459; Santa Cruz Biotechnology, Inc.), 100 nM miR-130b-3p inhibitor + 2 µl control-siRNA or 100 nM miR-130b-3p inhibitor + 2 µl PTEN-siRNA by using the Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells without any treatment were considered as the control group. The cells were subjected to the following experiments 48 h after transfection.

The transfection efficiency was detected 48 h after cell transfection, using RT-qPCR and/or western blot assay. Total RNA from tissues and cells were extracted using the TRIzol Reagent (Takara Bio, Inc.) according to the manufacturer's protocol. The RNA concentration was detected by NanoDrop spectrophotometer (ND-2000; Nanodrop Technologies; Thermo Fisher Scientific, Inc.). The total RNA was stored at −80°C until use. cDNAs were synthesized by using PrimeScript RT Reagent Kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol and analyzed by performing SYBR^® Premix Ex Taq™ II (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. The following primer sequences were used for qPCR: GAPDH forward, 5′-CGGGAAGCTTGTCATCAATGG-3′ and reverse, 5′-GGCAGTGATGGCATGGACTG-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; PTEN forward, 5′-GGAAAGGGACGAACTGGTGT-3′ and reverse-5′-CAGGTAACGGCTGAGGGAAC-3′; miR-130b-3p forward, 5′-CTGGTAGGGTACAGTACTGTGATA-3′ and reverse, 5′-CTGGTGTCGTGGAGTCGGC-3′. The thermocycling conditions were as follows: 95°C for 5 min, followed by 38 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 30 sec. GAPDH (for mRNA) and U6 (for miRNA) were used as the internal controls. Relative expression levels were calculated by the 2^−ΔΔCq method (18). All experiments were performed in triplicate.

Radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology) was used to extract proteins from cells or tissues according to the manufacturer's protocol. Bicinchoninic acid assay kit (Pierce Biotechnology; Thermo Fisher Scientific, Inc.) was used to quantify the protein samples in line with the manufacturer's protocol. The protein samples (25 µg/lane) were separated on 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk for 1.5 h at room temperature, followed by incubation with primary antibody against PTEN (cat. no. 9188; 1:5,000; Cell Signaling Technology Inc.), Akt (cat. no. 4691; 1:5,000; Cell Signaling Technology Inc.), phospho-Akt-B (p-Akt; cat. no. 4060; 1:5,000; Cell Signaling Technology Inc.), nuclear factor (NF)-κB-p65 subunit (p65; cat. no. 8242; 1:5,000; Cell Signaling Technology Inc.), phospho-NF-κB (p-NF-κB or p-p65; cat. no. 3033; 1:5,000; Cell Signaling Technology Inc.), survivin (cat. no. 2808; 1:5,000; Cell Signaling Technology Inc.), or β-actin (cat. no. 4970; 1:5,000; Cell Signaling Technology Inc.) overnight at 4°C. The membranes were then washed with Tris buffered saline with 0.1% Tween-20 (TBST) three times. Subsequently, the membranes were incubated with the anti-rabbit IgG, horse radish peroxidase-linked antibody (cat. no. 7074; 1:5,000; Cell Signaling Technology Inc.) for 2 h at room temperature. Finally, enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) was used to determine the immunoreactive bands. The band density was quantified with Gel-Pro Analyzer densitometry software (Version 6.3, Media Cybernetics, Inc.).

Cell proliferation was analyzed using the Cell Counting Kit-8 (CCK-8) assay (cat no. C0037; Beyotime Institute of Biotechnology). Logarithmic phase cells were seeded in a 96-well plate at a density of 1×10⁴ cells per well and incubated in the 37°C, 5% CO₂ incubator for 12 h. Subsequently, 10 µl CCK-8 was added to each well, and the cells were incubated for a further 2 h at 37°C with 5% CO₂. The absorbance was measured at a wavelength of 450 nm using a microplate reader.

WiT49 cells were transfected with either 100 nM inhibitor control, 100 nM miR-130b-3p inhibitor, 100 nM miR-130b-3p inhibitor + 2 µl control-siRNA or 100 nM miR-130b-3p inhibitor + 2 µl PTEN-siRNA for 48 h. Then, WiT49 cells were collected by 0.25% Trypsin. Apoptotic cells were detected by using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (cat. no. 70-AP101-100; MultiSciences) according to the manufacturer's protocol. Briefly, cells (10⁶) were incubated with 5 µl Annexin V-FITC and 5 µl PI for 30 min in the absence of light at room temperature. Finally, flow cytometry (BD Biosciences) was used to detect cell apoptosis. The data were analyzed using WinMDI software (version 2.5; Purdue University Cytometry Laboratories; www.cyto.purdue.edu/flowcyt/software/Catalog.htm).

TargetScan (www.targetscan.org) was used to predict the binding sites of miR-130b-3p and PTEN. To confirm the association between miR-130b-3p and PTEN, the dual-luciferase reporter vector pmiR-RB-REPORT™ (Guangzhou RiboBio Co., Ltd.) that contains a wild-type (WT) or mutant (MUT) 3′-UTR sequence of PTEN was constructed. Cells seeded in 24-well plates were co-transfected with miR-130b-3p mimics or mimic control and the MUT or WT 3′-UTR of PTEN using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After transfection for 48 h, cells were lysed with RIPA buffer. 48 h after transfection, the relative luciferase activity was detected using the Dual-Luciferase^® Reporter Assay System (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Firefly luciferase activity was normalized to that of Renilla luciferase.

All experiments were performed in triplicates. All data were shown as the mean ± standard deviation and SPSS v17.0 statistical software (SPSS, Inc.) was used for data analyses. Comparison between groups were performed by Student's t-tests and one-way analysis of variance followed by Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

To explore the role of miR-130b-3p in nephroblastoma, RT-qPCR was performed to detect the relative expression of miR-130b-3p in nephroblastoma (tumor tissues) of children with nephroblastoma. The results revealed that compared with the adjacent tissues, the expression of miR-130b-3p was significantly upregulated in nephroblastoma tissues in children with nephroblastoma (Fig. 1).

TargetScan analysis was performed to identify functional targets of miR-130b-3p. Hundreds of target genes were identified to be the potential target genes of miR-130b-3p and as shown in Fig. 2A, PTEN was one such target. Based on the literature analysis, it was identified that PTEN is one of the most common tumor suppressor in many human cancers and serves a vital role in the regulation of cell growth and apoptosis (16,19). Therefore, PTEN was selected for further investigations in the present study. Thus, to further examine whether miR-130b-3p directly targets PTEN, LucPTEN −3′UTR-WT and its 3′UTR-MUT plasmids were constructed. Luciferase reporter assay revealed that miR-130b-3p mimics clearly suppressed the luciferase activity of the WT-PTEN-3′UTR (Fig. 2B). Therefore, this provides evidence that PTEN is a direct target of miR-130b-3p.

In order to examine the expression of PTEN in nephroblastoma tissues, RT-qPCR and western blot analysis were performed. The results demonstrated that compared with the adjacent tissues, PTEN was less expressed in nephroblastoma tissues, at the mRNA and protein expression level (three representative cases in each group are shown; Fig. 3A-C) in children. The mRNA (Fig. 3D) and protein expression (Fig. 3E and F) of PTEN in WT-CLS1 and WiT49 cells was determined using RT-qPCR and western blotting, respectively. The results indicated that the mRNA (Fig. 3D) and protein expression (Fig. 3E and F) of PTEN was higher in WiT49 cells than in WT-CLS1 cells. WiT49 cells were then selected for further study.

WiT49 cells were transfected with inhibitor control or miR-130b-3p inhibitor for 48 h and RT-qPCR was performed to detect the efficiency of the inhibition. The results revealed that miR-130b-3p inhibitor significantly reduced miR-130b-3p expression in WiT49 cells (Fig. 4A). Furthermore, WiT49 cells were transfected with control-small interfering (si) RNA or PTEN-siRNA for 48 h, and subsequently, RT-qPCR and western blotting were performed to detect the efficiency of siRNA. The results demonstrated that PTEN-siRNA reduced both mRNA and protein levels of PTEN in WiT49 cells (Fig. 4B-D). Finally, in order to test whether miR-130b-3p had an effect on PTEN expression in WiT49 cells, WiT49 cells were transfected with: Inhibitor control, miR-130b-3p inhibitor, miR-130b-3p inhibitor + control-siRNA, or miR-130b-3p inhibitor + PTEN-siRNA for 48 h. RT-qPCR and western blotting were used to detect the expression of PTEN. It was revealed that miR-130b-3p inhibitor markedly increased PTEN expression levels at both mRNA and protein level, which was reversed by PTEN-siRNA (Fig. 4E-G).

In order to shed light on the function of miR-130b-3p in nephroblastoma, the effect of miR-130b-3p on the cell proliferation of WiT49 cells was investigated. The results demonstrated that miR-130b-3p inhibitor significantly decreased WiT49 cell viability, which was reversed by PTEN-siRNA (Fig. 5A). To further determine whether miR-130b-3p could regulate WiT49 cell apoptosis, flow cytometry was performed to analyze apoptosis. The results revealed that miR-130b-3p inhibitor significantly induced WiT49 cell apoptosis, and this induction was reversed by PTEN-siRNA (Fig. 5B and C).

Finally, to explore the molecular mechanism of the effect of miR-130b-3p inhibitor on nephroblastoma cells, Akt/p-NF-κB/survivin signaling pathway was analyzed. As shown in Fig. 6A, compared with the control group, the protein levels of p-Akt, p-NF-κB (p-p65) were markedly decreased by miR-130b-3p inhibitor, and these decreases were eliminated by PTEN silencing. The ratio of p-Akt/total-Akt and p-p65/p65 significantly decreased following treatment with the miR-130b-3p inhibitor, and these decreases were ameliorated following PTEN silencing (Fig. 6B and C). Furthermore, survivin protein levels were significantly decreased by miR-130b-3p inhibitor treatment. However, these decreases were reversed following PTEN silencing (Fig. 6D and E).