A total of 23 pairs of primary WT tissues (male, 12; female, 11; age, 3–60 months) and normal adjacent tissues (male, 13; female, 10; age, 8–64 months) were collected during therapeutic surgery at the Department of Surgery, Childrens' Hospital Affiliated to Soochow University, Soochow University (Suzhou, China). All of the specimens from the WT cases had a size of 1.0×1.0×0.2 cm and were pathologically diagnosed. Informed consent was obtained from all participants, and the present study was approved by the Institutional Review Board of the Childrens' Hospital Affiliated to Soochow University, Soochow University (Suzhou, China).

G401 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI 1640 medium (Gibco Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal calf serum (Invitrogen Life Technologies), 100 IU/ml penicillin and 100 mg/ml streptomycin (both from Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO_2. Plasmids encoding HDAC5-Flag, small interfering RNA (siRNA) specific for HDAC5 (sense 5′-CAUUGCCCACGAGUUCUCACCUGAU-3′ and anti-sense 5′-AUCAGGUGAGAACUCGUGGGCAAUG-3′) and siRNA specific for c-Met (sense 5′-AGCCAAUUUAUCAGGAGGUTT-3′ and antisense 5′-ACCUCCUGAUAAAUUGGCUTT-3′) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). HDAC5 and siRNA c-Met were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The cells were transfected using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. In brief, cells were seeded into six-well plates and transfected with 30 nM siRNA oligos with 4 µl Lipofectamine 2000 at 80% confluence.

The cells were seeded into 96-well plates at a density of 6.0×10³ cells/well. Cell viability was assessed using a CCK-8 assay (Beyotime Institute of Biotechnology, Haimen, China). The absorbance of each well was read using a spectrophotometer (4015-000; Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm.

BrdU incorporation assay was performed using a BrdU Cell Proliferation kit (Roche Diagnostics, Indianapolis, IN, USA) to analyze cell growth. Briefly, the cells were incubated with BrdU for 6 h, rinsed and then incubated with a fluorescein isothiocyanate-labeled antibody against BrdU for 30 min. The stained cells were then analyzed using a fluorescence micro-plate reader (2350; EMD Millipore, Billerica, MA, USA). Fold changes in BrdU incorporation were normalized against the mean fluorescence intensity of the control group.

Frozen tissues or cells were homogenized using TRIzol reagent (Invitrogen Life Technologies) and total RNA was isolated using a Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Reverse transcription was performed using a Takara RNA PCR kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's instructions. RT-qPCR was performed using SYBR Green Premix Ex Taq (Takara Bio, Inc.) on an ABI 7500 Cycling system (Applied Biosystems, Life Technologies, Thermo Fisher Scientific) to determine the expression levels of the genes of interest. The cycling parameters were as follows: Initial denaturation at 95°C for 30 sec, followed by a two-step program of 95°C for 5 sec and 60°C for 31 sec over 40 cycles. Experiments were performed in triplicate. The primers were obtained from Invitrogen Life Technologies and the sequences were as follows: HDAC5 forward, 5′-CTCAAGCAGCAGCAGCAGCTCCA-3′ and reverse, 5′-CCTTCTGTTTAAGCCTCGAACG-3′; c-Met forward, 5′-CCTTCGAAAGCAACCATTTTACG-3′ and reverse, 5′-TTACTGACATACGCGGCTTGCGC-3′. The expression levels were quantified using the 2^−∆∆Ct method.

Total protein was extracted with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) and quantified using the bicinchoninic acid assay (BAC kit; Beyotime Institute of Biotechnology). The proteins (20 µg) were fractionated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore). The membrane was blocked in 4% dried milk at room temperature for 1 h and incubated with primary antibodies at 4°C overnight. Rabbit anti-HDAC5 monoclonal antibody (cat no. 3443S; 1:1,000; Cell Signaling Technology, Inc., Beverly, MA, USA) and mouse anti-c-Met monoclonal antibody (cat no. sc-162; 1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used as primary antibodies. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (mouse-IgG; 1:5,000; Beyotime Institute of Biotechnology, Shanghai, China) for 1 h at room temperature and detected with an enhanced chemiluminescence system (Roche Diagnostics), according to the manufacturer's instructions. β-actin was purchased from Santa Cruz Biotechnology, Inc., which was used as a loading control.

Values are expressed as the mean ± standard deviation, based on three separate experiments. One-way analysis of variance was used for comparison between multiple groups and Student's t-test was used for comparison between two groups. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS, version 13.0 (SPSS, Inc., Chicago, IL, USA).

The mRNA expression levels of HDAC5 were examined in 23 paired WT and adjacent non-tumor tissues using RT-qPCR. It was observed that the expression of HDAC5 was significantly increased in the tumor tissues (Fig. 1A). In addition, the protein expression level of HDAC5 was determined using western blot analysis, which revealed increased expression levels of HDAC5 in the WT samples, compared with the adjacent normal samples (Fig. 1B). These data suggested that HDAC5 may be important in WT pathogenesis.

In order to elucidate the biological role of HDAC5 in WT, G401 cells were transfected with a recombinant plasmid containing HDAC5 (Fig. 2A). The subsequent CCK-8 assay demonstrated that overexpression of HDAC5 clearly promoted the proliferation of the WT cells (P<0.05) (Fig. 2B). BrdU incorporation is a standard method to measure DNA synthesis and is considered to be a surrogate procedure for the evaluation of proliferation (17). In the present study, the G401 cells overexpressing HDAC5 exhibited a faster growth rate, compared with the control G401 cells (Fig. 2C), consistent with the results of the CCK-8 assay. In addition, the G401 cells were transfected with siRNA targeting HDAC5, and the subsequent downregulation of the target gene was confirmed using RT-qPCR and western blotting (Fig. 3A and B). The data indicated that the downregulation of HDAC5 significantly decreased the rate of proliferation of the G401 cells (Fig. 3C and D). Taken together, these results indicated that HDAC5 may be a positive regulator of cellular proliferation in WT.

The present study also investigated the molecular mechanism underlying the proliferative effect of HDAC5, and the results demonstrated that the mRNA expression of c-Met was increased by >4-fold in the G401 cells overexpressing HDAC5, compared with the control G401 cells (Fig. 4A). Western blot analysis also confirmed the upregulation of c-Met in the HDAC5-transfected cells (Fig. 4B and C). By contrast, the expression of c-Met was reduced in the G401 cells following the downregulation of HDAC5 by siRNA interference (Fig. 4D–F). Taken together, these data suggested that HDAC5 was an upstream regulator of c-Met. In order to ascertain whether the induction of c-Met is a prerequisite for the proliferative function of HDAC5, the expression of c-Met was suppressed using siRNA oligos (Fig. 5A and B). Notably, the downregulation of c-Met inhibited the proliferative effects of HDAC5 on the G401 cells (Fig. 5C and D), suggesting that the proliferation-promoting function of HDAC5 was involved in the regulation of c-Met.