The expression profile of ECT2 in prostate cancer was examined using the University of Alabama at Birmingham Cancer data analysis (UALCAN) portal (http://ualcan.path.uab.edu) (16) database based on 52 adjacent normal tissues and 497 tumor samples.

The RWPE-1 human normal prostate epithelial cell line was purchased from American Type Culture Collection and cultured in keratinocyte serum-free medium (Invitrogen; Thermo Fisher Scientific, Inc.) with 0.05 mg/ml bovine pituitary extract and 5 ng/ml human recombinant epidermal growth factor. The LNCaP, DU145, PC-3 and 22RV1 human prostate cancer cell lines were obtained from Procell Life Science & Technology Co., Ltd. LNCaP and 22RV1 cells were incubated with RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin mixture (HyClone; Cytiva). DU145 cells were incubated with Minimum Essential Medium (Procell Life Science & Technology Co., Ltd.) with 10% FBS and 1% penicillin/streptomycin, while PC-3 cells were incubated with Ham's F-12K medium (Procell Life Science & Technology Co., Ltd.) in the presence of 10% FBS and 1% penicillin/streptomycin. All cells were cultured at 37°C in a humified incubator with 5% CO₂.

Total protein was isolated from PC-3 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was determined using a BCA kit (Beyotime Institute of Biotechnology). The same amounts of proteins (30 µg/lane) were separated by electrophoresis using a 12% SDS-PAGE gel, and then transferred to polyvinylidene fluoride membranes. Following blocking with 5% nonfat milk at room temperature for 2 h, the membranes were probed with primary antibodies against ECT2 (1:1,000; cat. no. ab86604; Abcam), MMP2 (1:1,000; cat. no. 10373-2-AP; Proteintech Group, Inc.), MMP9 (1:2,000, cat. no. 30592-1-AP; Proteintech Group, Inc.), E-cadherin (1:10,000; cat. no. ab40772; Abcam), N-cadherin (1:10,000; cat. no. ab76011; Abcam), Vimentin (1:1,000; cat. no. ab92547; Abcam), hexokinase 2 (HK2; 1:1,000; cat. no. ab209847; Abcam), pyruvate kinase M2 (PKM2; 1:1,000; cat. no. ab85555; Abcam), lactate dehydrogenase A (LDHA; 1:1,000; cat. no. ab5248; Abcam), ETS1 (1:1,000; cat. no. ab186844; Abcam) and GAPDH (1:2,500; cat. no. ab9485; Abcam) at 4°C overnight. The membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. ab6721; Abcam) for 2 h at room temperature. The blots were further developed using an enhanced chemiluminescence kit (Amersham; Cytiva) and semi-quantified using ImageJ software Version 1.52 (National Institutes of Health).

Total RNA was isolated from PC-3 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA concentration and purity were determined using a spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). Subsequently, 1 µg RNA was reverse-transcribed into cDNA using a First Strand cDNA Synthesis Kit (Sangon Biotech Co., Ltd.). The following conditions were used for RT: 42°C for 2 min, 37°C for 15 min and 85°C for 5 sec. Thereafter, SYBR Green qPCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for the qPCR assay according to the manufacturer's instructions. The qPCR thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 55°C for 5 sec and 72°C for 10 sec. The primer sequences used in the present study were as follows: ECT2 forward, 5′-ACTACTGGGAGGACTAGCTTG-3′ and reverse, 5′-CACTCTTGTTTCAATCTGAGGCA-3′; ETS1 forward, 5′-CCCGTACGTCCCCCACTCCT-3′ and reverse, 5′-TGGGACATCTGCACATTCCA-3′; and GAPDH forward, 5′-CAGGAGGCATTGCTGATGAT-3′ and reverse, 5′-GAAGGCTGGGGCTCATTT-3′. The mRNA levels of the genes were calculated using the 2^−∆∆Cq method (17). GAPDH was used as the internal control.

Short hairpin RNAs (shs) (pGPU6 vector) targeting ECT2 (sh-ECT2-1 sense, 5′-CCGGGCTGAGCATTCCCTTTCCATACTCGAGTATGGAAAGGGAATGCTCAGCTTTTTG-3′ and antisense, 5′-AATTCAAAAAGCTGAGCATTCCCTTTCCATACTCGAGTATGGAAAGGGAATGCTCAGC-3′; and sh-ECT2-2 sense, 5′-CCGGCGGAATGAACAGGATTTCTATCTCGAGATAGAAATCCTGTTCATTCCGTTTTTG-3′ and antisense, 5′-AATTCAAAAACGGAATGAACAGGATTTCTATCTCGAGATAGAAATCCTGTTCATTCCG-3′) and ETS1 (sh-ETS1-1 sense, 5′-CCGGGTGCAGATGTCCCACTATTAACTCGAGTTAATAGTGGGACATCTGCACTTTTTG-3′ and antisense, 5′-AATTCAAAAAGTGCAGATGTCCCACTATTAACTCGAGTTAATAGTGGGACATCTGCAC-3′; and sh-ETS1-2: sense, 5′-CCGGTGTGAAACCATATCAAGTTAACTCGAGTTAACTTGATATGGTTTCACATTTTTG-3′ and antisense, 5′-AATTCAAAAATGTGAAACCATATCAAGTTAACTCGAGTTAACTTGATATGGTTTCACA-3′) were constructed by Shanghai GenePharma Co., Ltd., with scrambled shRNA (pGPU6) as the negative control (sh-NC; sense, 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′ and antisense, 5′-AATTCAAAAACAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTG-3′). The Homo sapiens ETS1 full-length open reading frame was amplified and inserted into a pcDNA3.1 vector to construct an ETS1 overexpression vector (oe-ETS1; Shanghai GenePharma Co., Ltd.). Empty pcDNA3.1 vector served as the negative control (oe-NC; Shanghai GenePharma Co., Ltd.). shs plasmids (50 nM) and/or oe-ETS1/oe-NC (15 nM) were transfected into PC-3 cells using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for an incubation at 37°C for 6 h according to the manufacturer's instructions. PC-3 cells were not transfected served as the control. Following 48 h of culture at 37°C, the cells were harvested for subsequent experiments.

Cell proliferation was assessed using Cell Counting Kit-8 (CCK-8) and colony formation assays. For the CCK-8 assay, PC-3 cells were seeded into 96-well plates (1.0×10⁴ cells/well) and incubated in a 5% CO₂ incubator at 37°C for the indicated durations (24, 48 and 72 h). CCK-8 solution (10 µl; Dojindo Molecular Technologies, Inc.) was added to each well for an additional incubation at 37°C for 2 h. The absorbance at 450 nm was recorded using a microplate reader. The relative cell viability (%) was calculated using the following formula: (absorbance in treated group-absorbance blank)/(absorbance in control group at 24 h-absorbance blank) ×100.

In addition, PC-3 cells were seeded in 6-well plates (500 cells/well) and incubated in a 5% CO₂ incubator at 37°C for 2 weeks. The colonies (>50 cells) were fixed with methanol for 15 min at room temperature and stained with 0.1% crystal violet for 10 min at room temperature. Images were captured using light microscopy and colonies were observed by eye.

Cell migration was assessed using a wound healing assay. In brief, PC-3 cells were seeded in 6-well plates and incubated in a 5% CO₂ incubator at 37°C. When the cells reached 100% confluency, a single scratch wound was created using a 200-µl pipette tip. The plates were washed with PBS and subsequently cultured with serum-free medium for 24 h. Images were captured at 0 and 24 h using light microscopy. The cell migration rate (%) was calculated using the following formula: (0 h wound width −24 h width)/0 h wound width ×100.

Cell invasion was detected using an 8-µm pore Transwell chamber (Corning, Inc.) precoated with Matrigel (BD Biosciences) for 30 min at 37°C. A total of 2.0×10⁵ PC-3 cells were resuspended in serum-free medium and subsequently seeded into the upper Transwell chamber. The complete medium containing 10% FBS was plated in the lower chamber. Following incubation in a 5% CO₂ incubator at 37°C for 48 h, the non-invasive cells in the upper chamber were removed using a cotton swab. The invasive cells were fixed with methanol for 15 min at room temperature and stained with crystal violet for 10 min at room temperature. Finally, the images were observed using light microscopy and the invasive cells were counted using ImageJ software Version 1.52 (National Institutes of Health).

The cell culture medium was harvested and the lactate release and glucose uptake were evaluated using lactate assay (cat. no. MAK064) and glucose uptake assay kits (cat. no. MAK542) (Sigma-Aldrich; Merck KGaA), respectively, according to the manufacturer's guidelines.

In addition, cells (1.0×10⁴ cells/well) were seeded into XF96 cell culture microplates and incubated at 37°C overnight. The OCR and ECAR were detected using a Cell Mito Stress Test (cat. no. 103015-100) and Glycolysis Stress Test Kit (cat. no. 103020-100), respectively, on a Seahorse XFe96 Analyzer (all Agilent Technologies, Inc.), according to the manufacturer's guidelines.

The promoter region of ECT2 (−2,000 to transcription start site) or mutant promoter region of ECT2 without ETS1-binding sites was cloned into the pGL3-Basic vector (Promega Corporation) to construct the luciferase reporter vectors. PC-3 cells were co-transfected with the luciferase reporter vectors and oe-NC/oe-ETS1 using Lipofectamine 3000 reagent. Following cell culture for 48 h post-transfection, the luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega Corporation) according to the manufacturer's instructions, and normalized to Renilla luciferase activity.

The HumanTFDB (http://bioinfo.life.hust.edu.cn/HumanTFDB#!/) website was adopted to predict presumptive binding sites between the transcription factor ETS1 and the ECT2 promoter region, which was then verified using a ChIP assay, carried out using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, Inc.) according to the manufacturer's guidelines. In brief, PC-3 cells were fixed with 1% paraformaldehyde at 37°C for 10 min for crosslinking, quenched with 125 mM glycine at room temperature for 5 min, resuspended in SDS Lysis Buffer (MilliporeSigma) and subsequently sonicated into DNA fragments. After centrifugation at 12,000 × g for 5 min at 4°C, 100 µl of supernatant was incubated with 5 µg anti-ETS1 (cat. no. 14069; Cell Signaling Technology, Inc.) or anti-IgG (cat. no. 2729; Cell Signaling Technology, Inc.) antibodies. Protein A/G agarose beads (40 µl) (Santa Cruz Biotechnology, Inc.) were added to each IP reaction (100 µl) and incubated for 60 min at 4°C. The chromatin fragments were immunoprecipitated. After being washed with low-salt wash buffer, high-salt wash buffer and LiCI wash buffer, and being rinsed with TE buffer, the precipitated DNA was amplified and detected by qPCR as aforementioned.

Continuous variables are presented as the mean ± standard deviation from three repeats. Statistical analysis was conducted using an unpaired Student's t-test for two groups and one-way ANOVA with Tukey's post hoc test for more than two groups. The analysis was performed using GraphPad Prism software (version 8.0; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

Using the UALCAN database, it was determined that the expression levels of ECT2 were significantly upregulated in prostate cancer tumor samples compared with normal samples (Fig. 1A). Subsequently, the expression levels of ECT2 were examined in normal prostate epithelial cells and prostate cancer cells to confirm the abnormal ECT2 expression in prostate cancer. As shown in Fig. 1B and C, the mRNA and the protein expression levels of ECT2 in prostate cancer cell lines (LNCaP, DU145, PC-3 and 22RV1 cells) were considerably higher than those in RWPE-1 cells. Among these cell lines, ECT2 expression was highest in PC-3 cells. Therefore, PC-3 cells were used for subsequent experiments.

To explore the regulatory role of ECT2 in prostate cancer, loss-of-function experiments were performed. As shown in Fig. 2A and B, the expression levels of ECT2 were significantly downregulated following transfection with sh-ECT2-1/2. The sh-ECT2-1 vector (designated sh-ECT2 hereafter) was used in subsequent experiments due to its superior transfection efficacy. The subsequent cellular behavior assays revealed that knockdown of ECT2 expression could effectively inhibit the proliferation of prostate cancer cells, as shown by the reduced colony formation and cell viability in the sh-ECT2 group compared with the sh-NC group (Fig. 2C and D). Furthermore, wound healing and Transwell assays revealed that knockdown of ECT2 expression could reduce the wound closure and number of invasive cells, indicating decreased migration and invasion of PC-3 cells following ECT2 knockdown (Fig. 2E and F). Additionally, the inhibitory effect of knockdown of ECT2 expression on the expression levels of MMP2 and MMP9 (invasion-related proteins) in PC-3 cells further confirmed the anti-invasive effect of knockdown of ECT2 expression. In addition, the findings in Fig. 2G also revealed that knockdown of ECT2 expression significantly increased the expression levels of E-cadherin, a hallmark of epithelial cells, while significantly reducing the protein expression levels of N-cadherin and Vimentin (hallmarks of mesenchymal cells), suggesting that ECT2 knockdown in prostate cancer cells might retard the epithelial-mesenchymal transition which commonly occurs during cancer metastasis (18).

Prostate cancer cells can alter their glucose metabolism mode to aerobic glycolysis to meet the energy requirements for cell proliferation, migration, invasion and metastasis (19). Given that knockdown of ECT2 expression significantly inhibited the proliferation and invasion of PC-3 cells, additional experiments were performed to assess whether this effect was associated with changes in aerobic glycolysis. As shown in Fig. 3A and B, lactate release and glucose uptake were significantly decreased in the sh-ECT2 group compared with the sh-NC group. Accordingly, the decreased ECAR and increased OCR following sh-ECT2 transfection revealed that knockdown of ECT2 expression enhanced the glycolytic capacity, while it decreased ATP production and maximal respiration, suggesting that it impeded aerobic glycolysis in PC-3 cells (Fig. 3C). This was further verified by western blot analysis, which indicated that the expression levels of several critical enzymes in the aerobic glycolysis process, including LDHA, HK2 and PKM2, were significantly downregulated following knockdown of ECT2 expression in PC-3 cells (Fig. 3D).

Additional experiments were conducted to clarify the molecular mechanism of ECT2 and its regulation in prostate cancer cells. The HumanTFDB (http://bioinfo.life.hust.edu.cn/HumanTFDB#!/) website predicted that presumptive binding sites may exist between the transcription factor ETS1 and the ECT2 promoter region (Fig. 4A). It was observed that the expression levels of ETS1 were significantly upregulated in PC-3 cells compared with RWPE-1 cells (Fig. 4B and C). To explore the interaction between ETS1 and ECT2, PC-3 cells were transfected with oe-ETS1 plasmid to overexpress ETS1 or sh-ETS1-1/2 to interfere with ETS1 expression. sh-ETS1-1 was used for subsequent experiments due to its superior transfection efficacy (Fig. 4D and E). ETS1 overexpression significantly upregulated ECT2 expression, while knockdown of ETS1 expression significantly downregulated ECT2 expression (Fig. 4F and G), demonstrating that ETS1 could positively regulate ECT2. In addition, to confirm the binding site of ETS1 in the ECT2 promoter, luciferase reporter and ChIP assays were conducted. The data revealed that the luciferase activity in cells co-transfected with ECT2-wide-type (ECT2-WT) and oe-ETS1 was markedly increased in comparison to that in cells co-transfected with ECT2-WT and oe-NC. There was no difference of the luciferase activity in cells with ECT2-mutant-type (Fig. 4H). Furthermore, the enrichment of precipitated chromatin fragments containing binding sites to the ECT2 promoter in the anti-ETS1 group was significantly higher than that in the IgG group (Fig. 4I). Therefore, these data confirmed that ETS1 could directly bind to the ECT2 promoter and positively regulate ECT2 expression at the transcriptional level in PC-3 cells.

To verify the involvement of ETS1 underlying ECT2-mediated prostate cancer progression, gain- and loss-of-function experiments were conducted in PC-3 cells. As shown in Fig. 5A, PC-3 cells were transfected with sh-ECT2/sh-NC alone or co-transfected with sh-ECT2 and oe-ETS1/oe-NC, and the CCK-8 assay revealed that knockdown of ECT2 expression inhibited cell viability, which was partly reversed by additional ETS1 overexpression. Knockdown of ECT2 expression-caused reduction in colonies was partly abolished following ETS1 overexpression (Fig. 5B). Subsequently, wound healing and Transwell assays revealed that the inhibitory effects of knockdown of ECT2 expression on cell migration and invasion were partly reversed by ETS1 overexpression (Fig. 5C and D). Furthermore, simultaneous transfection with sh-ECT2 and oe-ETS1 significantly increased the protein expression levels of MMP2, MMP9, N-cadherin and Vimentin and decreased the protein expression levels of E-cadherin compared with those following transfection with sh-ECT2 and oe-NC (Fig. 5E). In addition, simultaneous transfection of the cells with sh-ECT2 and oe-ETS1 significantly increased lactate release and glucose uptake compared with transfection with sh-ECT2 and oe-NC. This was accompanied by upregulated ECAR and downregulated OCR, revealing that the inhibitory effect of knockdown of ECT2 expression on aerobic glycolysis was partly weakened by ETS1 overexpression (Fig. 6A-C). Furthermore, ECT2 knockdown-reduced protein expression levels of HK2, PKM2 and LDHA were partly restored by additional ETS1 overexpression (Fig. 6D).