The human RCC cell lines KETR3 and 786-O were obtained from the Cell Bank of Chinese Academy of Sciences and cultured in DMEM (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (penicillin, 100 U/ml; streptomycin, 0.1 mg/ml; Sigma-Aldrich; Merck KGaA) at 37°C with 5% CO₂. In total, ~6×10⁴ cells were cultured overnight until 60–70% convergence and cells were subsequently transfected with 50 nM small interfering RNA (siRNA/si)-DLEU1 (Oligobio) or scrambled siRNA [as negative control (NC); Oligobio] using Lipo6000 (Beyotime Institute of Biotechnology) and cultured for 6–8 h at 37°C. Subsequently, the medium containing the transfection reagent was removed and the cells were cultured in DMEM for 24–48 h before the next experiments. The sequence of siRNA-DLEU1 was 5′-CAACGGAAUGUAUCAAUGATT-3′, the sequence of siRNA-control was 5′-TTCTCCGAACGTGTCACGT-3′.

A total of 24 h post-transfection, total RNA was isolated from transfected cells using an Ultrapure RNA kit (CoWin Biosciences Co., Ltd.) and reverse transcribed into cDNA using a HiFiScript cDNA Synthesis kit (CoWin Biosciences Co., Ltd.) according to the manufacturer's protocol. qPCR was performed with a SYBR Premix Ex Taq II kit (Takara Bio, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 3 sec and 60°C for 30 sec. Data were analyzed according to the sample quantification cycle (Cq) value from three independent experiments. The relative gene expression was calculated using the 2^−∆∆Cq method (25). Target gene expression was normalized to the expression of β-actin. The primer sequences were as follows: DLEU1, forward 5′-CCAGCCCACAGGCATTTAGT-3′, reverse, 5′-GTTCCGAGGCTTAAGTGCGA-3′; and β-actin, forward 5′-CCCGAGCCGTGTTTCCT-3′ and reverse, 5′-GTCCCAGTTGGTGACGATGC-3′.

A CCK-8 (Beijing Solarbio Science & Technology, Co., Ltd.) assay was conducted to assess cell viability. Briefly, cells were seeded in each well of a 96-well plate at a density of 1×10³ cells/well; after transfection for 0, 24, 48 and 72 h, cells were treated with CCK-8 reagent (10 µl) for an additional 1 h at 37°C. The optical density value of excitation was detected using a microplate reader (BioTek Instruments, Inc.) at a wavelength of 450 nm. The assay was conducted three times independently.

A total of 24 h post-transfection, cells were seeded in a 6-cm dish at a density of 200 cells/well and were cultured for ~1 week at 37°C in an atmosphere containing 5% CO₂ until visible colonies were formed. The culture medium was removed, and 5 ml 4% paraformaldehyde was added to fix the colonies for 30 min at room temperature followed by staining with 0.1% crystal violet for 30 min at room temperature. The colonies were counted under a light microscope (Nikon Corporation, Inc.) and images were captured. The assay was conducted three times independently.

Transwell chambers with 8-µm pore filters (EMD Millipore) coated with Matrigel (BD Biosciences) in a 24-well plate were used for the cell invasion assay; chambers were not coated with Matrigel for the migration assay. A total of 24 h post-transfection, cells were trypsinized and resuspended in serum-free culture medium at a concentration of 1×10⁶ cells/ml. Subsequently, a 100- or 5-µl cell suspension was added to the upper compartment of Transwell chambers for the invasion and migration assays, respectively. Complete medium (600 µl) with 10% FBS was added to the bottom chamber to serve as a chemoattractant. After 48 h at 37°C, the residual cells on the upper surface were removed, and the cells that had invaded or migrated to the lower surface of the filter were fixed with 4% paraformaldehyde for 30 min at room temperature, followed by staining with 0.1% crystal violet for 20 min at room temperature. Images of the invaded and migrated cells were captured (magnification, ×40) and counted under a light microscope. The assay was conducted three times independently.

Cells transfected with siRNA for 24 h were resuspended in 1X binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl₂] at a density of 1–5×10⁶ cells/ml. Subsequently, the cell suspension (100 µl) was stained with 5 µl Annexin V-FITC (BD Biosciences) in the dark for 5 min at room temperature and incubated with 10 µl propidium iodide (PI) for 5 min at room temperature. The rate of apoptosis was analyzed by flow cytometry (BD FACSCanto II; BD Biosciences) and calculated using BD FACSDiva™ software (version 6.0; BD Biosciences). The assay was conducted three times independently.

After 48 h of transfection, cells were harvested and lysed using RIPA lysis buffer (CoWin Biosciences Co., Ltd.) at 4°C for protein extraction. A bicinchoninic assay kit (Beyotime Institute of Biotechnology) was used to measure the protein concentration according to the manufacturer's protocol. Proteins (20 µg/sample) were separated via 10% SDS-PAGE and were then transferred onto PVDF membranes (EMD Millipore), which were blocked with 5% non-fat milk for 1 h at room temperature, followed by incubation overnight at 4°C with primary antibodies (1:1,000). After washing with TBS-Tween-20 (20%), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies anti-rabbit immunoglobulin G (IgG; 1:3,000; cat. no. SA00001-2; ProteinTech Group, Inc.) or anti-mouse IgG (1:3,000; cat. no. SA00001-1; ProteinTech Group, Inc.) for 1 h at room temperature. An enhanced chemiluminescence kit (CoWin Biosciences Co., Ltd.) was used for signal development, and the bands were analyzed using ImageJ software version 1.44 (National Institutes of Health). The primary antibodies used were as follows: Anti- Bcl-2 (cat. no. 60178-1-Ig; ProteinTech Group, Inc.), Bax (cat. no. 50599-2-Ig; ProteinTech Group, Inc.), total caspase-3 (cat. no. 66470-2-Ig; ProteinTech Group, Inc.), cleaved caspase-3 (cat. no. 25546-1-AP; ProteinTech Group, Inc.), total caspase-9 (cat. no.9502; Cell Signaling Technology, Inc.), cleaved caspase-9 (cat. no. 10380-1-AP; ProteinTech Group, Inc.), Akt (cat. no. 9272; Cell Signaling Technology, Inc.), phosphorylated (p)-Akt (cat. no. 66444-1-Ig; ProteinTech Group, Inc.), cyclin D1 (cat. no. 60186-1-Ig; ProteinTech Group, Inc.), P70S6K (cat. no. 14485-1-AP; ProteinTech Group, Inc.) and GAPDH (cat. no. cat. no. 60004-1-Ig; ProteinTech Group, Inc.). The assay was conducted three times independently.

SPSS 18.0 software (SPSS, Inc.) was used for statistical analysis. The data from triplicate experiments are presented as the means ± standard deviation. The difference between two groups was compared with Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the functional role of DLEU1 in the growth of RCC cells, loss-of-function experiments were performed in the RCC cell lines KETR3 and 786-O using si-DLEU1; scrambled siRNA was used as the NC. As presented in Fig. 1A and B, the expression levels of DLEU1 were significantly downregulated in si-DLEU1-transfected cells compared with in NC cells (P<0.05). A CCK-8 assay was then performed using cells transfected with si-DLEU1. Notably, the growth curves of KETR3 cells demonstrated a significant reduction in the viability of si-DLEU1-transfected cells compared with NC cells (P<0.05; Fig. 1C). Additionally, 786-O cells transfected with si-DLEU1 exhibited a significant suppression of cell viability (P<0.05; Fig. 1D). Furthermore, a colony forming assay further revealed that DLEU1 knockdown significantly decreased the number of KETR3 and 786-O cell colonies compared with in the NC group (P<0.05; Fig. 1E and F), thus suggesting that DLEU1 knockdown inhibited the clonogenic ability of RCC cells.

To determine the functional role of DLEU1 in the progression of RCC, Transwell assays were performed to detect the migration and invasion of KETR3 and 786-O cells. As presented in Fig. 2A, si-DLEU1 transfection significantly decreased the ability of KETR3 and 786-O cells to migrate into the lower chamber compared with NC cells (P<0.05). Additionally, compared with the NC, si-DLEU1-transfected cells exhibited a significant reduction in their ability to invade through the Matrigel into the lower chamber (P<0.05; Fig. 2B). Collectively, these data indicated that DLEU1 knockdown reduced the migratory and invasive abilities of RCC cells in vitro.

To further assess the effect of DLEU1 on the survival of RCC cells, cell apoptosis was evaluated using flow cytometry. It was demonstrated that DLEU1 knockdown significantly increased the percentage of apoptotic KETR3 cells compared with in the NC group (P<0.05; Fig. 3A). Similarly, silencing DLEU1 with siRNA significantly promoted apoptosis of 786-O cells (P<0.05; Fig. 3A). Subsequently, the expression of apoptosis-associated proteins was evaluated to further investigate the mechanisms underlying the increased apoptosis induced by the downregulation of DLEU1 in RCC cells. From the results of western blot analysis, it was revealed that DLEU1 downregulation significantly inhibited the expression of Bcl-2, total caspase-3 and total caspase-9, and increased the expression of Bax, cleaved caspase-3 and cleaved caspase-9 in KETR3 and 786-O cells compared with in the NC group (P<0.05; Fig. 3B). Collectively, these findings suggested that the increase in apoptosis induced by DLEU1 knockdown may be dependent on its regulation of the Bcl-2/Bax axis and caspase cascade in RCC cells.

The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is an established pathway involved in the occurrence and development of cancer (26). To determine whether the Akt signaling pathway was involved in the function of DLEU1, the phosphorylation levels of Akt, and the expression of downstream proteins cyclin D1 and P70S6 kinase (P70S6K) were evaluated via western blot analysis. It was observed that silencing DLEU1 significantly reduced the phosphorylation levels of Akt in KETR3 and 786-O cells compared with the NC (P<0.05), but induced no notable effects on the expression of total Akt (Fig. 4A). Additionally, the expression levels of cyclin D1 and P70S6K, proteins involved in the cell cycle, were similarly downregulated in DLEU1 knockdown cells (P<0.05; Fig. 4A).

Due to the critical role of EMT in promoting tumor cell migration and invasion, the expression of EMT marker proteins was further assessed to investigate whether EMT was involved in the function of DLEU1. It was revealed that si-DLEU1-transfected KETR3 cells demonstrated a significant increase in the expression of E-cadherin, an important marker of epithelial cells, with similar observations in 786-O cells (P<0.05; Fig. 4B). Furthermore, DLEU1 knockdown also induced a significant downregulation in the expression of N-cadherin and vimentin, which are important mesenchymal markers, in KETR3 and 786-O cells (P<0.05; Fig. 4B). Collectively, these results suggested that DLEU1 knockdown interfered with the mesenchymal properties of RCC cells.