Fifty-eight human RCC tissue samples and adjacent normal tissues were obtained from RCC patients (49 males and 9 females; age, 46–70 years with mean of 58.6 years) undergoing nephrectomy at the Department of Urology, Renmin Hospital of Wuhan University (Wuhan, China). All of the tumor samples were of the clear-cell sub-type as verified by pathological examination, and the corresponding normal samples obtained from adjacent tissues did not exhibit any pathological abnormalities. The samples were frozen in liquid nitrogen and stored at −70°C prior to protein and RNA extraction. The use of human tissues was approved by the Institutional Review Board of Wuhan University (Wuhan, China) and conformed to the Declaration of Helsinki. All of the patients provided written informed consent.

The 786-0 and HK-2 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The Caki-1 and Caki-2 cell lines were purchased from the China Center for Type Culture Collection (Wuhan, China). All cell lines were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% (v/v) penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere containing 5% CO₂.

FOXO4 overexpression plasmids were constructed with pcDNA3.1/Flag (Clontech Laboratories, Inc., Mountainview, CA, USA) as the vector, FOXO4 as the expression gene and flag as a tag. The primer sequences for FOXO4 were as follows: Sense, 5′-ATGGATCCGGGAATGAGAAT-3′ and anti-sense, 5′-TCAGGGATCTTGGCTCAAAG-3′. The constructed vector was verified using standard DNA sequencing using an ABI PRISM 310 Genetic Analyzer (PerkinElmer, Inc., Waltham, MA, USA). An empty expression plasmid was used as a control. A total of 1×10⁵ cells were seeded into each well of a six-well plate. Upon 70% confluency, the medium was discarded and replaced with fresh non-supplemented, serum-free medium. A total of 1.5 µg pcDNA3.1/Flag/FOXO4 plasmid or pcDNA3.1/Flag was mixed with 100 µl Opti-MEM (Invitrogen; Thermo Fisher Scientific, Inc.). Furthermore, 4.5 µl Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was mixed with 100 µl Opti-MEM and incubated for 5 min at room temperature. Subsequently, the plasmid and lipofectamine 2000 solutions were mixed and incubated for another 20 min prior to addition to the cells. Following 24 h of incubation, the transfection efficiency was determined by western blot analysis and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analyses.

Bim siRNA and scrambled control siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Cells were transfected with siRNA following the manufacturer's instructions. In brief, 2×10⁵ 786-0 cells were cultured in 2 ml antibiotic-free medium in a six-well plate and transfected with pcDNA3.1/Flag/FOXO4 24 h prior to treatment with Bim siRNA. First, 2–8 µl of siRNA duplex was added to 100 µl siRNA transfection medium (solution A) and 2–8 µl of siRNA transfection reagent was added to 100 µl siRNA transfection medium (solution B). Subsequently, solutions A and B were mixed and incubated at room temperate for 30 min. Following washing of each well with 2 ml siRNA transfection medium, 0.8 ml siRNA transfection medium was added to each well containing the siRNA transfection reagent mixture (200 µl from solutions A and B). After incubation for 8 h, the supernatant was removed and replaced with normal medium, followed by incubation for another 24 h prior to analysis. The silencing efficiency was examined using western blot and RT-qPCR. All assays, including transfections, western blots and PCR, were performed as at least three independent experiments.

ccRCC cells were transfected with pcDNA3.1/Flag/FOXO4 or pcDNA3.1/Flag and incubated for 24 h. Subsequently, all cultured cells and the media were collected, and following centrifugation at 1,000 × g for 5 min, cells were re-suspended in 300 µl ice-cold binding buffer. Cells were then stained with 10 µl propidium iodide (PI) and 5 µl Annexin V-fluorescein isothiocyanate (FITC) using an Annexin V-FITC kit (Beyotime Institute of Biotechnology, Haimen, China) and incubated for another 5 min. Ten thousand events were analyzed to determine the proportion of apoptotic cells using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Tissue and cell extracts were obtained using radioimmunoprecipitation assay lysis buffer (Biyuntian Biotechnology Co., Shanghai, China) on ice and the lysates were centrifuged for 15 min (12,000 × g, 4°C). Protein concentration was determined using the QuantiPro™ BCA assay kit (Sigma-Aldrich, St. Louis, MO, USA). Proteins (1 µg/µl; 20 µl) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Shanghai Haling Biotechnology Co., Ltd., Shanghai, China) and subsequently electroblotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking non-specific binding to the membrane with 5% non-fat milk, the membranes were incubated overnight at 4°C with the following primary antibodies at a 1:1,000 dilution: Rabbit monoclonal anti-FOXO4 (Abcam, Cambridge, MA, USA; cat. no. ab128908), rabbit polyclonal anti-Bim (Abcam; cat. no. ab7888), rabbit polyclonal anti-B-cell lymphoma 2 (Bcl-2; Abcam; cat. no. ab59348), rabbit monoclonal anti-Bcl-2-associated X (Bax; Abcam; cat. no. ab32503), rabbit monoclonal anti-cleaved-caspase 3 (Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 9664), rabbit polyclonal anti-cytochrome c (Abcam; cat. no. ab90529) and rabbit polyclonal anti-β-actin (Santa Cruz Biotechnology, Inc.; cat. no. sc-130657). Membranes were washed three times with Tris-buffered saline with Tween 20 (TBST; Wuhan Guge Biotechnology Co., Ltd, Wuhan, China) and incubated with corresponding IRDye horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (LI-COR Biosciences, Lincoln, NE, USA; cat. no. 925-32211) for 2 h at room temperature. The membranes were rinsed three times with TBST and the protein bands were visualized using a two-color infrared imaging system (Odyssey; LI-COR Biosciences).

Total RNA was isolated from the kidney tissues and cultured cells with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using the PrimeScript™ RT reagent kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's instructions. RNA (1.5 µg) extracted from tissues was used as a template to perform one-step RT-PCR, and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The resulting cDNA was amplified using real-time PCR in a volume of 20 µl with the SYBR Green Master mix (Takara Bio, Inc.) method and the ABI 7500 Real-Time RT-PCR system (Thermo Fisher Scientific, Inc.). The conditions of the PCR were initial denaturation at 95°C for 30 sec, followed by 40 cycles of 5 sec at 95°C, 30 sec at 60°C and 1 min at 72°C. The Cq values of each samples were calculated using the 2^−ΔΔCq data analysis method (13). The primer sequences were generated by Sangon Biotech Co., Ltd. (Shanghai, China), as follows: GAPDH forward, 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse, 5′-AGGGGCCATCCACAGTCTTC-3′; and FOXO4 forward, 5′-CTTTCTGAAGACTGGCAGGAATGTG-3′ and reverse, 5′-GATCTAGGTCTATGATCGCGGCAG-3′.

Experiments were repeated three times independently and representative data are shown. Values are expressed as the mean ± standard error of the mean. Student's t-test was used for comparison between two groups, and one-way analysis of variance was used for comparisons between groups. P<0.05 was considered to indicate a statistically significant difference between values.

In order to examine expression levels of FOXO4, 58 clinical renal cancer samples were obtained and analyzed using RT-qPCR. As shown in Fig. 1A, FOXO4 expression was significantly reduced in 51 (87.9% of total) human RCC tissues compared with that in their corresponding adjacent non-tumorous tissues. Furthermore, the FOXO4 protein expression was assessed in 13 pairs of randomly selected RCC tissues and their adjacent normal tissues using western blot analysis. As shown in Fig. 1B and C, the protein levels of FOXO4 were significantly decreased in RCC tissues compared with those in their corresponding normal tissues in 11 out of 13 pairs (P<0.05). In addition, as shown in Fig. 1D and E, the relative expression levels of FOXO4 mRNA and protein in the three RCC cell lines 786-0, Caki-1 and Caki-2 were significantly reduced compared to those in the HK2 normal human renal tubular cell line.

In order to clarify the role of FOXO4 in ccRCC cells, two ccRCC cell lines were transfected with pcDNA3.1/FOXO4, which resulted in high expression of FOXO4. As shown in Fig. 2, western blot and RT-qPCR indicated that in 786-0 and Caki-1 cells, FOXO4 protein and mRNA expression in the pcDNA3.1/FOXO4 plasmid group was upregulated compared with that in the empty plasmid group.

Following transfection with pcDNA3.1/FOXO4 plasmid or pcDNA3.1 plasmid for 24 h, the apoptotic rate was assessed by FACS using Annexin V/PI double staining. The results revealed that overexpression of FOXO4 significantly increased the apoptotic rate of 786-0 (25.0±8.0) and Caki-1 (15.9±4.1) cells, while the apoptotic rate of the control-transfected cells was 3.1±0.2 in 786-0 cells and 1.3±0.2 in Caki-1 cells (Fig. 3A). To identify the mechanism of FOXO4-induced apoptosis in 786-0 cells, FOXO4-overexpressing cells were subjected to western blot analysis of apoptosis-associated proteins at 24 h following transfection. As shown in Fig. 3B, Bax, cleaved-caspase 3, cytochrome c and Bim were upregulated and Bcl-2 was downregulated in 786-0 and Caki-1 cells transfected with pcDNA3.1/FOXO4.

In order to assess the role of Bim in FOXO4-induced apoptosis, Bim siRNA was used to suppress the expression of Bim in FOXO4-transfected 786-0 cells or control-transfected cells. As shown in Fig. 4A, western blot analysis indicated that Bim was downregulated after siRNA treatment. Of note, knockdown of Bim expression led to a substantial decrease in the apoptotic rate of FOXO4-overexpressing 786-0 cells (P<0.05) (Fig. 4B and C). In addition, the expression of apoptosis-associated proteins was altered following knockdown of Bim. In the pcDNA3.1-FOXO4 + Bim-siRNA group, cleaved-caspase 3, Bax and cytochrome c were significantly downregulated compared with those in the pcDNA3.1-FOXO4 group. However, knockdown of Bim was not able to rescue the levels of Bcl-2, which were significantly downregulated in the pcDNA3.1-FOXO4 group (Fig. 4D).