We speculated on the differential expression of AOC1 and its relationship with SOX15 using seven Gene Expression Omnibus (GEO) datasets (GSE46602, GSE71016, GSE28204, GSE27616, GSE38241, GSE114740, and GSE6752; https://www.ncbi.nlm.nih.gov/geo). Based on these GEO datasets, we analyzed differentially expressed genes of prostate cancer and normal prostate tissue using the online program GE02R based on the limma R package (http://www.ncbi.nlm.nih.gov/geo/geo2r/). False-positive data were amended by adjusting the p-value. Hence, we chose adjusted p < 0.05 and log FC > 1 or log FC < −1 as the significant data. Venn diagrams and volcano plots were used to distinguish the intersection of differentially expressed genes across these GEO datasets. To optimize the relationship between the expression of AOC1 and SOX15 and the prognostic value of prostate cancer, we supplemented the data with the Cancer Genome Atlas (TCGA) cohort, which was analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/). We obtained the protein–protein interaction networks of AOC1-encoded proteins (amiloride-binding protein 1) using the Search Tool for Recurring Instances of Neighboring Genes (STRING) (https://string-db.org/). Furthermore, we predicted the transcription factor of the AOC1 promoter from the JASPAR CORE database (http://jaspar.genereg.net/).
Ninety-four tissue samples were collected from prostate cancer patients between 2011 and 2013, at the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China). The patients in this study received a definite diagnosis by pathology examination and had not undergone medical treatment. Written informed consent was obtained from every patient included in the study. The research was approved by the Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University (ZZU-LAC20210924 (15)).
Cell culture and transfection
Human prostate cancer cell lines DU145 and 22Rv1 were purchased from the American Type Culture Collection (LGC Standards, London, England). Both cell lines were periodically certified and tested for mycoplasma contamination. The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 1% penicillin–streptomycin liquid (Gibco, Carlsbad, CA, USA) at 37 °C with 5% CO2. Cells were cultured to 60–80% confluence; then, they were transfected using jetPRIME® in vitro DNA and siRNA transfection reagent (Polyplus-transfection, Strasbourg, France) according to the manufacturer’s instructions.
Quantitative real-time polymerase chain reaction
Total RNA was extracted using the TRIzol® reagent (Invitrogen). The cDNA was prepared with NovoScript® Plus All-in-one 1st Strand cDNA Synthesis SuperMix (gDNA Purge). (Novoprotein, Shanghai, China). Gene transcripts were quantitated using the QuantStudio Three Real-Time PCR System (Thermo Fisher) using the NovoStart® SYBR qPCR SuperMix Plus (Novoprotein), with GAPDH as an internal control. The primers sequence used during our quantitative real-time polymerase chain reaction (qRT-PCR) test is shown in Supplementary Table 1.
Paraffin-embedded tissues were sliced into 5-μm sections and observed for AOC1 expression after immunohistochemistry staining. These sections were dyed with diluted anti-AOC1 antibody (1:500; ab231558; Abcam, Cambridge, UK) and incubated at 4 °C overnight; then, they were washed three times with fresh phosphate-buffered saline (PBS) solutions and immediately incubated with biotin-labeled second antibody for immunohistochemistry (GB23303; Servicebio, Wuhan, China) for 50 min at room temperature. The positive cells were monitored using diaminobenzidine solution (K5007; DAKO, Santa Clara, CA, USA) by direct viewing. The processed slides were observed using a light microscope (Leica DM2700). Every immunohistochemistry result was read blindly and independently by two senior pathologists.
The positive cell scores were defined as follows: 1 point, 1–9% positive cells; 2 points, 10–50% positive cells; 3 points, 51–80% positive cells; and 4 points, >80% positive cells. We defined the staining intensity scores as follows: 1 point, negative staining; 2 points, weak staining; 3 points, moderate staining; and 4 points, strong staining. Our final immunoreactivity score was the product of the positive cell score and staining intensity score.
The total protein content in tissues and cells was extracted using RIPA buffer (Solarbio, Beijing, China) supplemented with 1% PMSF (Solarbio). The quantification was completed with the help of the BCA Protein Assay Kit (Solarbio). We used SDS–PAGE gels prepared from the PAGE Gel Fast Preparation Kit (Epizyme, shanghai, China) to separate the total protein (20 μg). Isolates were immediately anchored onto nitrocellulose membranes (Millipore, Danvers, MA, USA). A blocking solution for 1 h at room temperature was necessary for more specific antibody binding. The blotted nitrocellulose membranes were incubated with the primary antibody, anti-AOC1 antibody (1:1000; ab231558; Abcam, UK), anti-TFR antibody (1:1000; ab214039; Abcam), anti-TF antibody (1:1000; ab277635; Abcam,), anti-FTH1 antibody (1:1000; ab75973, Abcam), and anti-SOX15 antibody (1:100; sc-166964; Santa Cruz, Dallas, TX) overnight at 4 °C. After washing with Tris-buffered saline with Tween, the nitrocellulose membranes were incubated with goat anti-rabbit IgG (IRDye® 800CW; 1:5000; ab216773; Abcam) or goat anti-mouse IgG (IRDye® 800CW). Anti-GAPDH antibody (1:1000; ab9485; Abcam) was used as a loading control. The Odyssey CLx Infrared Imaging System (Gene Company Limited, Hong Kong, China) was used to detect the target protein bands.
Cell counting kit-8 assay
The cell counting kit-8 (CCK8) assay (Dojindo, Tokyo, Japan) was used to assess the proliferative viability of tumor cell lines. Three thousand cells were cultured in 96-well plates filled with 100 μL RPMI-1640 and 10% FBS for 96 h. Then, 10 µL of the CCK8 reagent was supplemented to each well 4 h after cell placement. The absorbance in each well was measured with a DNM-9606 microplate reader (Perlong, Beijing, China) at 450 nm every 24 h.
We used the EdU assay kit (RiboBio, Guangzhou, China) according to the manufacturer’s protocol to verify the proliferation abilities of the cells. The treated cells were placed in 96‐well plates at 4 × 104 cells/well overnight. Then, cells were treated with 50 μmol/L EdU and incubated for 6 h at 37 °C, followed by fixation with paraformaldehyde solution for 15 min and permeabilization with 0.5% Triton X‐100 for 20 min. Incubation with 100 μL of 1 × Apollo® reaction cocktail performed for 30 min at room temperature and protection from light was the most important conditions. Finally, the nuclei of the cells were dyed with DIPA for 5 min and observed by fluorescence microscopy.
Colony formation assay
Approximately 1200 22Rv1 and DU145 cells were placed on the plate. The RPMI-1640 with 10% FBS was replaced every 5 days with a fresh medium. After culturing for 15 days, the cells were fixed with 1 mL of paraformaldehyde for 30 min; then, they were washed three times with PBS. Dyeing with 0.1% crystal violet for 30 min was the most important step. The clones were conscientiously counted after being washed and dried at room temperature.
A wound-healing assay was used to confirm the migration ability of cell lines. Altogether, 1 × 106 prostate cancer cells were placed and cultured in RPMI-1640 with FBS. When the cell density was 80–100%, a wound was made in the cell monolayer, and the prime image was immediately photographed. Then, cells placed in six-well plates were cultured for another 12 or 48 h, and the corresponding images were photographed immediately. The area of cell migration was our primary outcome measure used to analyze the migration ability of cell lines.
The cell migration ability was also assessed using the Transwell assay with 8-μm pores (Corning Inc., Corning, NY, USA) as a supplement. The cells were collected by trypsin digestion and placed into the upper chambers with 1 × 105 cells; then, the medium in each well was replenished to 200 μL. The upper chambers were placed in a 24-well plate after each well of RPMI-1640 medium with 20% FBS. After being incubated for 24 h at 37 °C and 5% CO2, the upper chambers were stained, observed, and analyzed.
The content of H2O2 in the cells was analyzed using a content detection kit (Solarbio, Beijing, China). The cells were collected in a centrifuge tube, and the supernatant was discarded after centrifugation; 1 mL reagent I was added per 5 × 106 cells, and cells were broken using ultrasonic waves (power, 20%; ultrasonic waves, 3 s; interval, 10 s; repetitions, 30) followed by centrifugation at 8000×g for 10 min at 4 °C. Finally, the supernatant was placed on ice before testing. The spectrophotometer or microplate reader was preheated for more than 30 min and the wavelength was set to 415 nm; it was adjusted to zero using distilled water. Reagents II, III, and IV were placed in a water bath at 37 °C for more than 10 min. Samples and reagents were added in order according to the instruction for use and allowed to stand at room temperature for 5 min; then, 200 μL was transferred to a 96-well plate to determine the absorbance at 415 nm and calculate ∆A, from which the H2O2 content was calculated.
ROS fluorescence probe assay
The H2DCFDA (DCFH-DA, DCFH) ROS fluorescence probe (MKBio, Shanghai, China) was used to complement and validate the content of intracellular ROS. A total of 2.5 × 105 cells to be tested in each well were placed in a six-well plate, and the culture medium was extracted before adding the 10 mM of working solution with PBS. They were incubated at room temperature for 40 min. The staining working solution was sucked out and washed once with preheated cell culture medium. The pre-warmed cell culture medium was added again before evaluation using a microscope.
A micro-malondialdehyde (MDA) assay kit (Solarbio) was purchased to detect the level of lipid oxidation in cell membranes. The cells were collected in a centrifuge tube, and the supernatant was discarded after centrifugation; 1 mL of reagent I was added per 5 million cells. Ultrasonic waves (power, 20%; ultrasonic waves, 3 s; interval, 10 s; repetitions, 30 times) were used to break the bacteria or cells before centrifugation at 8000 × g for 10 min at 4 °C; the supernatant was collected and placed on ice before testing. The spectrophotometer or microplate reader was preheated for more than 30 min and the wavelength was set to 415 nm; this was adjusted to zero with distilled water. The samples and reagents I, II, and III were added in order according to the IFU. After the mixture was kept warm in a water bath at 100 °C for 60 min (capped tightly to prevent water loss), it was cooled in an ice bath at room temperature and centrifuged at 10,000 × g for 10 min.
A 200-μL aliquot of the supernatant was pipetted into a 96-well plate to measure the absorbance at 450, 532, and 600 nm to determine the ΔA, from which the MDA content was calculated.
Liperfluo fluorescence probe assay
The Liperfluo fluorescence probe (Dojindo, Beijing, China) was used to complement and validate the intracellular lipid peroxide content. A total of 2.5 × 105 cells to be tested in each well were placed in a six-well plate, and the culture medium was extracted before adding the 10 mM of working solution with PBS and incubating at room temperature for 40 min. The staining working solution was sucked out and washed once with preheated cell culture medium. The cells were resuspended with pancreatin and later detected using a flow cytometer (excitation wavelength, 488 nm; emission wavelength, 515–545 nm).
In vivo tumor xenograft model
According to the requirements of statistical methods, we randomly selected 5 mice in each group for the experiment, which is also consistent with most articles. The severe combined immunodeficiency mice (4–5 weeks old; male) used for in vivo transplantation experiments were purchased from Sibeifu Company (Beijing, China). The 22Rv1 cells (6 × 106) infected with vector and shControl, AOC1 and shControl, and AOC1 and siSOX15 were dispersed in 50 μL PBS and 50 μL Matrigel (Corning) and inoculated subcutaneously into the dorsal flank of the mice. A power analysis was performed to calculate the sample size needed for our experiments. All animals were randomly assigned to different groups. Observation and measurement of the tumor size were performed every 3 days, and the tumor was removed and weighed on day 33.
Experimental procedures were performed in accordance with the guidelines established by the National Institutes of Health of China and approved by the Ethics Committee for Animal Experiments of the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
All data were analyzed using GraphPad Prism 7 Software (GraphPad, San Diego, CA, USA) and SPSS v. 24.0 software (2010; IBM, Chicago, IL, USA). Unless otherwise stated, two-tailed Student’s t-test or an analysis of variance was used to assess significance. Three replicates were performed for every experiment. P < 0.05 indicated statistical difference.