Patients and specimens
A total of 57 prostate cancer clinical tissue samples were used in this study; they were obtained from the First Affiliated Hospital of China Medical University. The Gleason score was determined at the Department of Pathology, the First Affiliated Hospital of China Medical University, and the samples were collected between 2013 and 2017. This study was approved by the Ethics Committee of China Medical University, and all patients signed informed consent forms.
In vivo nude mouse experiments
The experimental protocol for the animal experiments in this study was approved by the Institutional Animal Care and Use Committee of China Medical University (CMU2021546) and adhered to the guidelines for the care and use of laboratory animals issued by the China Animal Research Council. Four-week-old female BALB/c nude mice were purchased from Charles River and housed in specific pathogen-free (SPF) “barrier” facilities. We injected 1 × 107 PC3 cells stably overexpressing ACAT1 under the right creaking fossa of each female mouse. The mice were raised for 1 month. During this period, SPF mice were provided chow and allowed to drink sterile water ad libitum. The temperature was maintained at 22–26 °C and suitable humidity was maintained under a 12−/12-h light/dark cycle. After the end of the experiment (1 month), the tumor was excised from each mouse, the weight and size of the tumors were measured, and statistical analysis was performed.
All cell lines were obtained from the Shanghai Cell Bank (Shanghai, China) and cultured in RPMI 1640 medium with fetal bovine serum (FBS; FB15015; Clark Biosciences, Richmond, VA, USA). LNCaP and PC3 cells were cultured in a medium containing 10% FBS and no antibiotics, according to the manufacturer’s instructions, and maintained in a 5% CO2 incubator at 37 °C.
Plasmid construction and transfection
ACAT1 siRNA (siB170726094953-1-5) and normal control cells were purchased from Ruibo Biosciences (Guangzhou, China). The overexpression plasmid ACAT1-3Flag of ACAT1 and its control GV141 were obtained from GENE (Shanghai, China). MAP 1LC3B-Flag (RC207356) was obtained from Origene (Beijing, China). The cells were transfected using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Immunohistochemistry and Gleason scores
The 57 tissue sections were incubated with ACAT1 rabbit polyclonal antibody (16215-AP-1, 1:100; ProteinTech Group, Rosemont, IL, USA) at 4 °C overnight. Thereafter, they were incubated with the secondary antibody for 2 h at 37 °C. The nuclei were stained with hematoxylin for 10 min; 100 μL of 3,3′-diaminobenzidine chromogenic solution (P0202, Beyotime Biotechnology, Shanghai, China) was added to each tissue section and ACAT1 expression was observed under a microscope. Next, the tissue sections were strictly scored according to the latest Gleason scoring system. The χ2 test was used to determine the correlation between ACAT1 expression and clinicopathological characteristics. P < 0.05 was considered to indicate a statistically significant difference.
The total cell protein was extracted using lysis buffer (P0013; Beyotime Biosciences, Shanghai, China). We added a mixture of protease and phosphatase inhibitors (B14002 and B15002; Biotool, Shanghai, China), four times the volume of the cell pellet, to the cell lysate. The total protein (35 μg) was separated via SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (EMD-Millipore, Billerica, MA, USA). The membrane was blocked with 5% skimmed milk (232,100; BD Biosciences, Franklin Lakes, NJ, USA) for at least 2 h and then incubated with the appropriate primary antibody overnight at 4 °C. Peroxidase-conjugated secondary antibody was added and incubated at 37 °C for 1 h. The following primary antibodies were used: Anti-ACAT1 (16215-AP-1, 1:1000), anti-P62/SQSTM1 (66184-1-lg, 1:1000), anti-NRF2 (16396-1-AP, 1:1000), anti-lamin B1 (66095-1-lg, 1:1000), anti-FUS (60160-1-lg, 1:1000), and anti-GAPDH (60004-1-Ig, 1:1000) from ProteinTech Group, and anti-LC3B (3868 s, 1:1000) from Cell Signaling Technology (Denver, MA, USA). The total protein was visualized using an enhanced chemiluminescence method (34,080; Thermo Fisher Scientific, Waltham, MA, USA). Image J software (National Institutes of Health, Bethesda, MD, USA) was used to evaluate each band quantitatively and Prism 5.0 software (GraphPad, La Jolla, CA, USA) was used to compare the signal intensity.
Quantitative real-time polymerase chain reaction (q-PCR)
q-PCR was performed using SYBR Green PCR Master Mix in a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with a total reaction volume of 20 μL. The q-PCR conditions were as follows: 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 30 s, for 40 cycles. The dissociation step was used to generate a melting curve and confirm amplification specificity. The expression level relative to β-actin expression was calculated using the 2-ΔΔCt method.
The cell lines used in the experiment were seeded in two 10-cm cell culture dishes. When the cells reached confluence, they were lysed for 20 min and centrifuged at 12000 rpm for 15 min at 4 °C. Next, 40 μL of protein A/G Sepharose (P2012; Beyotime Biosciences) was added to the supernatant and blocked for at least 2 h. The mixture was then centrifuged at 1000 rpm for 5 min at 4 °C, and the supernatant was divided into two parts. Anti-ACAT1 or anti-FUS antibody (8 μg) was added to one part, and anti-mouse/rabbit IgG (1:2000; ZSGB-BIO, Beijing, China) to the other part. The mixture was shaken overnight at 4 °C. The next day, 25 μL of agarose A/G magnetic beads was added to each tube and incubated at 4 °C for 6 h. The cell lysate was then washed, heated in boiling water for 10 min, and finally, immunoblotting was performed.
Immunofluorescence co-localization experiments were performed with PC3 and LNCaP cell lines. The two prostate cancer cell lines were fixed in a glass-bottomed dish with paraformaldehyde, and then anti-ACAT1 (1:50) and anti-FUS (1:50) antibodies were added and incubated for 16 h. The next day, the secondary antibody was added and incubated for 2 h at 37 °C, the nucleus was stained with DAPI, and finally, the image was acquired using a laser confocal microscope (FV3000, Olympus, Tokyo, Japan).
The target cells were collected and separated from the cytoplasm and nucleoproteins. The experiment was then performed according to the instructions provided by the manufacturer, using a nuclear and cytoplasmic protein extraction kit (P0027, Beyotime Biosciences). The separated proteins were then boiled for 5 min and subjected to western blotting.
EdU cell proliferation assays
LNCap or PC3 cells stably transfected with FUS or ACAT1 were added to a 35-mm cell culture dish, and after overnight culture, the corresponding reagents were added according to the instructions of Edu (C0075L; Beyotime Biotechnology, Shanghai, China), and finally photographed using a confocal laser microscope and statistical analysis was performed.
Transwell cell migration experiment
Stably transfected cell lines were seeded (5 × 104 cells) into Transwell chambers (Costar, Washington, DC, USA). The Transwell chambers were plated with Matrigel (Corning Life Sciences, Corning, NY, USA), 1 day in advance, and then the Transwell chambers with 5 × 104 cells were placed in a 24-well culture plate with 600 μL of FBS for 24 h. After 24 h, the Transwell chambers were washed thrice with pre-cooled PBS, the cells were fixed with pre-cooled methanol for 10 min, and then washed an additional three times with PBS. After washing, the cells were stained with crystal violet for 10 min. Micrographs were captured using CellScan software, and the cells were counted using PS software. Finally, GraphPad Prism 5 was used to analyze the obtained data. Statistical significance was set at P < 0.05. This experiment was performed in triplicates.
Active oxygen detection assays
The level of reactive oxygen species (ROS) generated by prostate cancer cells was detected using the probe 2′,7′-dichlorodihydrofluorescein (DCFH-DA, Beyotime Biotechnology), which detected diacetate. After 48 h of transfection with the SIRT5 plasmid or siRNA, the cells were cultured in the dark for 20 min with 10 μM DCFH-DA in a humidified atmosphere at 37 °C in the presence of 5% CO2. The cells were washed three times with cold phosphate-buffered saline to remove excess fluorescent probes. The cells were counted, 104 cells were added to each well of a 96-well plate, and the absorbance was measured at 488 nm.
Chromatin immunoprecipitation (ChIP)
For ChIP, 2 × 107 PC3 and LNCaP cells overexpressing LC3B were used. The experiment was performed using the Millipore ChIP kit (17-371, Boston, MA, USA) according to the manufacturer’s instructions. Finally, micrographs were obtained using Image Lab software. The antibody used was anti-Flag (66008-4-lg, ProteinTech). The primers used were FUS forward, 5′-CGTCCCCAGCCGCCGGGACCG-3′ and reverse, 5′-ACGCTCCGCCGCCTACGCACCG-3′.
In this study, we used Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/) and STRING: functional protein association networks (https://string-db.org/). The GEPIA database was used to analyze ACAT1 expression in and prognosis of prostate cancer, and the STRING database was used to analyze the interaction between proteins.
All data were analyzed using SPSS version 24.0 (Beijing, China) to perform the χ2 tests, and Prism 5 (GraphPad) software was used for ROS analysis and signal intensity analysis in western blotting experiments. All experiments were repeated at least three times independently under the same conditions. Results with P < 0.05 were considered statistically significant.