Cell culture
The PCa cell lines LNCaP, C4-2, PC3, DU145, C42R (20 μm of Enzalutamide induced for 120 days) and 22Rv1 were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 medium with 10% fetal bovine serum (Gibco Cat# 10099141 C) at 37 °C and 5% CO2. All the cell cultures contained 1% penicillin and 1% streptomycin (Gibco Cat# 1037801). When the cell density reached 80-90%, 0.25% trypsin was used for sub-culturing.
Chronic virus transfection assays
The day before transfection, 22RV1 cells with good cell status were inoculated in the 6-well plate, and the confluent degree reached 30-50% the next day. Transfection was performed with a special transfection reagent polybrene, followed by a system of 2 mL culture medium per well, 2.5 µL polybrene and 10 µL disease venom. Fresh culture medium was replaced 4–6 h after transfection. After 48 h, the transfection efficiency was verified by RT-PCR and Western blot. When the mRNA and protein expression levels of JARID1D were significantly reduced, it was confirmed that the stable lentivirus strain was successfully constructed.
RT-PCR assays
A total RNA extraction kit was used to extract the total RNA of each group of cell samples, and a reverse transcription kit was used to reverse-transcribe RNA into cDNA. The reaction conditions were 37 °C for 15 min and 85 °C for 15 s. Then real-time fluorescent quantitative PCR was conducted on the cDNA of each group of samples. With β-Actin is an internal reference gene, Formula 2−ΔΔCT calculates the relative expression levels of each group of genes. The reagents and instruments used were PrimeScript™ RT Reagent Kit, TB Green® Fast qPCR Mix (Takara, Beijing, China), Step One Plus real-time PCR Detection System (Applied Biosystems, Thermo Fisher, Waltham, MA, USA). PCR reaction conditions were set as follows: predenaturation at 95 °C for 30 s, PCR at 95 °C for 15 s, and PCR at 60 °C for 30 s for 40 cycles. The primer sequences used for PCR are listed in Supplementary Table 1.
Western blot analysis
According to the experimental protocol reported in the literature [12], the protein was extracted from the cells with lysate, and the protein concentration was determined with BCA protein quantitative kit. Then, 10% SDS-PAGE gel was assembled, gel electrophoresis was performed, and proteins were separated according to molecular weight. Electrophoresis conditions: 90 V, 400 mA, 120 min. The transfer box was assembled with the gel closest to the negative electrode and the film closest to the positive electrode. Transfer condition: 90 V, 400 mA, time depends on the size of the molecular weight. After the transfer, the PVDF membrane was soaked in 5% skim milk powder for 2 h. The membrane was incubated with a primary antibody against the target protein, and shaken gently overnight at 4 °C, or incubated at room temperature for 1 h. Finally, an imaging system was used to capture and analyze the images, and the protein antibodies used in the experiment were: JARID1D (Affinity Biosciences, DF2548, 1: 1000), AR (Abcam, ab108341, 1:1000), N-cadherin (United Kingdom, Abcam, ab76011, 1:1000), E-cadherin (Proteintech, 20874-1-AP, 1:1000), MMP2 (United Kingdom, Abcam, ab92536, 1:1000), H3K4me3 (Affinity, DF6935, 1:1000), Vimentin (Proteintech 10366-1-AP, 1:1000) and β-actin (Engibody, AT0001, 1:2000).
Transwell assay
The 22RV1 sh-JARID1D cells were established via the transfection of chronic viruses to knock down JARID1D expression; 22RV1-NC cells were the corresponding controls. These cells were added to the chamber and cultured for 48 h. The cells were then fixed with 4% paraformaldehyde and stained with crystal violet, and the microscope was used to capture the images of the cells passing through the Transwell chamber.
Animal experiment
Male BALB/c nude mice, aged 6–7 weeks, were sourced from GemPharmatech Co. Ltd. (Chengdu, China) and housed in a pathogen-free environment at the Laboratory Animal Center of the Fourth Military Medical University (FMMU). Our animal study protocol was meticulously reviewed and approved by the Institutional Review Board of the Institutional Animal Care and Use Committee of FMMU (Protocol code: No. 20200417, approved on March 20, 2020), ensuring all procedures adhered to the highest ethical standards.
In our study, 2 × 106 22RV1 sh-JARID1D and 2 × 106 22RV1-NC cells were injected into the tail veins of the mice. To monitor metastasis in vivo, small-animal optical imaging was employed using the Caliper Lumina II system after a 4-week observation period. Lung tissue sections were subsequently harvested for histological examination via H&E staining upon detection of metastatic signals.
For the curcumin intervention experiments, 2 × 106 22RV1 sh-JARID1D cells labeled with luciferase were similarly injected. After a 4-week establishment period, mice were randomly assigned to two groups (n = 7 per group) in a blinded manner, ensuring equal distribution of baseline characteristics. The treatment group received intraperitoneal injections of curcumin (Selleck, USA, S1848) at a dosage of 30 mg/kg body weight, administered biweekly, while the control group received equivalent volumes of the vehicle. Body weight was closely monitored throughout the study to assess general health. Bioluminescence imaging was conducted weekly for 3 weeks using the Xenogen IVIS50 imaging system, with the following parameters: field of view set to ‘D’, binning at ‘4’ for optimal signal-to-noise ratio, exposure time adjusted based on initial pilot scans to maximize the dynamic range without saturation, and f/stop at ‘1’ for maximum light collection. Signal quantification was performed using Living Image 4.7.3 software, applying a standardized protocol for region of interest (ROI) identification and quantification. We defined metastatic signals as any ROI with a total flux exceeding a threshold of 1 × 106 photons/second. Upon completion of the study, mice were humanely euthanized, and lung tissues were collected for paraformaldehyde fixation, ensuring rigorous adherence to our predefined monitoring protocols and ethical considerations.
Methylation specific PCR (MSP)
Genomic DNA was extracted using proteinase K digestion and phenol/chloroform extraction, which was quantified using an ultraviolet spectrophotometer and stored at − 20 °C. DNA was purified using a Wizard clean-up system kit (Takara, Beijing, China), and the specific steps were carried out according to the manufacturer’s instructions. DNA bisulfite modification was performed using standard protocols [13]. The detailed procedure involved initial denaturation at 99 °C for 10 min, followed by incubation at 50 °C for 60 min with intermittent mixing, and a final cooling step at room temperature for 10 min. After 24 h of culture, the methylation status of the cells in each group was detected by the MSP method [14]. The MSP primer sequences, annealing temperatures, and fragment sizes of JARID1D are listed in Supplementary Table 2.
Cell viability analysis
According to the product manual provided by the manufacturer, cell samples were collected and Cell Count Kit-8 (7 Sea Biotech, Shanghai, China) was used to determine their growth activity. Tumor cells were cultured in 96-well plates (3 × 103 cells/well), and curcumin was added at varying concentrations. Following a 72-hour incubation, 100 µL medium-CCK-8 mixture at a ratio of 9:1 was added and incubated for 2 h. Absorbance values were measured at 450 nm, and cell viability was calculated based on these values. Finally, use GraphPad software, select normalized Response Variable slope, perform curve fitting, and obtain its IC50 value. The relevant data are listed in Supplementary Table 3.
ChIP assay
ChIP assays were determined with the Simple ChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, 91820). A total of 7.5 × 106 cells were cross-linked at room temperature for 10 min using a 1% solution of paraformaldehyde. The cross-linking reaction was quenched by adding 1 mL of 1.25 M glycine and incubating for 5 min. The cross-linked chromatin was subsequently transferred to AFA fiber tubes (Covaris) and sonicated to achieve an average fragment size of 150–200 base pairs (bp) in a 0.2% SDS shearing buffer using a Covaris sonicator. The sonicated chromatin was centrifuged at 13,000 rpm for 5 min and diluted to a final concentration of 0.1% SDS. The chromatin was pre-cleared using Dynabeads Protein G (Life Technologies) for 1 h. Chromatin-protein complexes were then immunoprecipitated with 5 μm of specific antibodies at 4 °C overnight. On the subsequent day, Dynabeads Protein G were added again for a 2-hour incubation. The beads were thoroughly washed with a series of buffers: low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, and finally with TE buffer. The precipitated chromatin was eluted from the beads using 300 µL of elution buffer (1% SDS, 0.1 M NaHCO3) for 1 h at room temperature, followed by de-cross-linking at 65 °C overnight. The ChIP and input DNA samples were treated with RNase A and proteinase K, and then extracted using phenol-chloroform extraction. The integrity and size of the DNA fragments (150–200 bp) were assessed using a High Sensitivity DNA Kit on an Agilent 2100 Bioanalyzer. The antibodies used in the experiment were: anti-AR (Cell Signaling Technology, 5153), anti-JARID1D (Affinity Biosciences, DF2548), and anti-H3K4me3 (Abcam, ab6002) antibodies. In addition, anti-histone H3 antibodies (Cell Signaling Technology, 4620) and normal rabbit IgG antibodies (Cell Information Technology, 2729) were used as positive and negative control groups, respectively. Finally, to analyze the DNA derived from ChIP, quantitative RT-PCR was used. For the quantitative RT-PCR analysis, we used SYBR Green Master Mix and specific primers designed to amplify ROIs. The PCR program included an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Data were normalized to input DNA and expressed as fold enrichment relative to a negative control region. The relevant primers are listed in Supplementary Table 1.
Co-immunoprecipitation (Co-IP)
For Co-IP, we prepared 8 × 106 cells and lysed them in lysis buffer containing 10 mM Hepes-NaOH (pH 8), 1.5 mM MgCl2, 25% glycerol, 0.5% Nonidet P-40, 0.42 M NaCl, 0.2 mM EDTA, and 0.5 mM DTT. After centrifugation at 13,200 rpm for 10 min, diluted in a dilution buffer (20 mM Tris-HCl, pH 8.0, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5% Nonidet P-40), and supplemented with 3 mM MgCl2 and 20 U/mL DNase (NEB). The mixture was incubated at 37 °C for 30 min. To preclear the lysate, 30 µL of Dynabeads Protein G (Life Technologies) were added and incubated at 4 °C for 60 min with gentle rotation. Subsequently, 10% of the lysate was reserved as the input control, and the remainder was incubated with 5 µg of the following primary antibodies overnight at 4 °C: JARID1D (Affinity Biosciences, DF2548, 5 µg/µL), AR (Cell Signaling Technology, 5153, 5 µg/µL), IgG (SC-2027, 2 µg/µL). On the following day, 50 µL of Dynabeads Protein G were added to the lysate and incubated for 2 h at 4 °C with gentle rotation. The precipitated proteins were then washed twice with a low-salt Co-IP wash buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1.5 mM MgCl2, 0.5% Nonidet P-40, 0.2 mM EDTA). Finally, 30 µL of NuPAGE LDS sample buffer with DTT (Bio-Rad) was added to the beads, and the mixture was heated at 95 °C for 5 min. The supernatants were then subjected to immunoblotting to detect the proteins of interest.
Immunohistochemistry (IHC)
The process began with tissue sectioning and preparation, followed by deparaffinization and antigen retrieval to expose antigenic sites. Endogenous peroxidase was blocked, and non-specific binding was prevented with serum blocking. The sections were then incubated with a primary antibody to target the protein of interest, followed by an enzyme-conjugated secondary antibody. After washing, a chromogenic substrate was applied for signal development, typically resulting in a colored reaction product at the antigen location. The sections were counterstained, dehydrated, and mounted for microscopic analysis to assess protein presence and distribution. IHC analysis was performed using antibodies against JARID1D (USA, Affinity Biosciences, DF2548, 1:100), AR (United Kingdom, Abcam, ab108341, 1:200), E-cadherin (Proteintech, 20874-1-AP, 1:5000), N-cadherin (United Kingdom, Abcam, ab76011, 1:200), MMP2 (United Kingdom, Abcam, ab92536, 1:100), Vimentin (Proteintech, 10366-1-AP, 1:3000) and CK8 (United Kingdom, Abcam, ab53280, 1:250).
H&E staining
H&E staining is a commonly used histological technique in pathology for evaluating the morphological characteristics of tissue sections. In our study, tissue sections were first subjected to fixation, followed by hydration. They were then stained with hematoxylin to color the cell nuclei. After staining, the sections were rinsed with water and subsequently stained with eosin to color the cytoplasm and other tissue structures. Once the staining process was complete, the sections were dehydrated through a series of gradually increasing concentrations of alcohol, ultimately clearing away excess dye and undergoing clarification treatment. Finally, the sections were mounted in a resin medium to protect the staining and were observed and analyzed under a light microscope.
Statistical analysis
The data are presented as mean ± standard error (SE), and were analyzed using GraphPad Prism (GraphPad Software, San Diego, USA). Differences between experimental groups were tested using the Student’s t-test and one-way and two-way analysis of variance (ANOVA) (P < 0.05).
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