Patient samples and cell culture
All patients provided informed consent in accordance with the Ethics Committee of Shanghai Jiao Tong University School of Medicine and the Renji Hospital Ethics Committee (KY2022 136A). Detailed patient information is listed in Supplementary Table S1. All cell lines were obtained from the Shanghai Chinese Academy of Sciences (Shanghai, China). PCa cell lines, including PC3, DU145, and 22RV1, were cultivated in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Yesean, Shanghai, China). Human normal prostate epithelial cells (RWPE-1) were cultured in a Defined Keratinocyte SFM medium (Gibco). PCSCs were cultured in serum-free DMEM/F12 medium (Gibco) supplemented with 20 ng/ml epidermal growth factor (EGF, Sigma, Saint Louis, USA), 20 ng/ml basic fibroblast growth factor (bFGF, Sigma), 0.4% bovine serum albumin (BSA, Sigma), 5 μg/ml insulin (Sigma), and N2 supplement (Stemcell Technologies Inc., Canada) as previously described [34].
Western blots
PCa cells were lysed in RIPA lysis buffer for 30 minutes on ice, and protein concentrations were measured using the BCA Protein Assay (Beyotime Biotechnology, Shanghai, China). Each sample contained 30 μg of protein, loaded onto SDS-PAGE gels (Bio-Rad Laboratories) and transferred to PVDF membranes (Sigma-Aldrich). Following a 1-hour block with 5% BSA at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies, listed in Supplementary Table S2, included CD133, CD44, SOX2, KLF4, TSG101, ALIX, CD9, Calnexin, METTL13, USP10, AKT, p-AKT, ERK1/2, p-ERK1/2, NFκB, p-NFκB, and GAPDH. After extensive washing with PBST, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Subsequent washes with PBST preceded chemiluminescence detection (Cell Signaling Technology) using the Odyssey two-color infrared laser imaging system (LI-COR Biosciences, Lincoln, NE, USA).
RNA isolation and real-time PCR
Total RNA was extracted from PCa cells using Trizol reagent (Yesean, China). Reverse transcription of cDNA was performed with the HiScript® III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China), and real-time PCR was conducted using the ChamQ SYBR qPCR Master Mix Kit (Vazyme, China) according to the manufacturer’s instructions. Primers for the genes were synthesized by Sangon (Shanghai, China), with detailed information listed in Supplementary Table S2.
Isolation and identification exosomes derived from PCSCs (PCSC-exos)
The conditioned medium of PCSCs, cultured for 48 hours, was collected and centrifuged at 3000 ×g for 15 minutes to remove dead cells and debris. After further centrifugation at 10,000 ×g for 30 minutes to remove extracellular vesicles (EVs), the supernatant was filtered through 0.22 µm filter membranes and ultracentrifuged at 100,000 ×g at 4°C for 1 hour. The pellet was then resuspended in PBS for exosome research [35]. Transmission electron microscopy (TEM) analyzed the size distribution of exosomes. The BCA Protein Assay Kit (Vazyme, China) quantified the protein concentration of exosomes. Exosomal markers TSG101, CD9, and ALIX, along with the negative marker calnexin, were detected via western blot. Additionally, CM-Dil dye (Yesean, China) labeled exosomes as described previously [36]. Briefly, PCa cells were incubated with CM-Dil-labeled exosome solution at 4°C in the dark for 15 minutes. After washing three times with PBS, imaging analysis was performed using confocal laser scanning microscopy (TCS SP5, Leica).
Lentivirus infection and cell transfection
TcDNA3.1-based vectors encoding the full-length cDNA of human AGD1 and METTL13, as well as control plasmids and short hairpin sequences against AGD1, METTL13, and a negative control, were constructed by Genomeditech (Shanghai, China). Detailed information about these plasmids is listed in Supplementary Table S2. Briefly, these plasmids were transfected into HEK-293T cells, and the resulting virus was used to infect PCa cells. The treated cells were selected with puromycin to obtain stable cell lines with gene knockdown or overexpression.
For cell transfection, Lipofectamine 3000 (Invitrogen, USA) was used to transfect the overexpression or shRNA plasmids into PCa cells according to the manufacturer’s instructions. Real-time PCR and western blot analyses were performed to evaluate the efficiency of overexpression or knockdown.
CCK-8, EdU incorporation, colony formation, and flow cytometry assays
Cell proliferation was assessed using the CCK-8 kit (Yesean, Shanghai, China) and 5-Ethynyl-2′-Deoxyuridine (EdU) assay (Beyotime Biotechnology, China) following treatment with 20 nM docetaxel (Meilune, Dalian, China), according to the manufacturer’s instructions.
For the colony formation assay, 1,000 cells were seeded per well in 6-well plates and cultured for 14 days. The cells were then fixed with 75% ethanol and stained with 0.1% crystal violet, with images captured using a digital camera.
The Annexin V-FITC/PI assay was conducted using the Annexin V-FITC/PI KIT (Becton, Dickinson and Company, #556547, USA). Stemness of PCa cells was analyzed using the ALDEFLUOR™ assay kit (StemCell Technologies, Herndon, VA, USA). Treated cells were harvested, thoroughly washed with PBS, and incubated with the appropriate dye per the manufacturer’s instructions. After another PBS wash, the cells were analyzed on an LSRFortessa SORP (BD Biosciences, Franklin Lakes, NJ), and data were processed with FlowJo software (Ashland, OR). Three independent experiments were performed.
Animal assays
Four-week-old male BALB/C nude mice, purchased from SLAC Laboratory Animal (Shanghai, China), were maintained under SPF conditions. Mice were randomly assigned to control and treatment groups. In the subcutaneous tumor experiment, 5 × 106 PCa cells in 100 μL were injected subcutaneously into the right inguinal region. After one week, docetaxel (10 mg/kg) was administered via intraperitoneal injection twice weekly, with tumor sizes measured twice weekly using the formula: volume = 0.5 × length × width2. After one month, mice were sacrificed, and tumors were measured and fixed in 4% paraformaldehyde. For the pulmonary metastatic assay, 1 × 106 luciferase-expressing PCa cells in 100 μL were injected into the tail vein of six-week-old nude mice. Docetaxel (10 mg/kg) was administered intraperitoneally twice weekly starting one week post-injection. After one month, mice were monitored weekly using an in vivo imaging system (IVIS), and metastatic foci were analyzed using aniView software. All animal experiments adhered to the Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011) and were approved by the Animal Care Committee of Shanghai Jiao Tong University School of Medicine.
Prostasphere formation
A total of 1,000 treated PCa cells per well were resuspended in 500 μL of DMEM/F12 medium supplemented with growth factors and cultured in 24-well ultra-low-attachment plates. The medium was refreshed every 2-3 days. After two weeks, the prostaspheres were measured and photographed using light microscopy. Prostaspheres with diameters greater than 100 μm were considered stem colonies. Three independent experiments were conducted.
Organoid culture and lentiviral transduction
Organoids derived from patients with CRPC exhibiting docetaxel resistance were established based on previous reports [37, 38]. Lentiviral transduction of shRNA AGD1 and shRNA NC followed the previously described protocol [39]. Briefly, organoids were trypsinized, suspended in lentivirus-containing organoid media with 8 μg/mL polybrene, and incubated for 1 hour at 37°C before embedding in Matrigel. After 48-72 hours of culture, organoids were selected with 1 μg/mL puromycin. The proliferation of organoids treated with docetaxel was analyzed on the fifth day using the CCK-8 assay. Three independent experiments were conducted.
RNA pull-down assay, RNA immunoprecipitation (RIP) assay, and co-immunoprecipitation (Co-IP) assay
An AGD1 pull-down assay was performed using an RNA pull-down kit (Bersinbio, Guangzhou, China) following the manufacturer’s instructions. AGD1 and control probes were designed and synthesized by GenePharma (Shanghai, China), with detailed information provided in Supplementary Table S2. Briefly, 107 PCa cells were lysed on ice for 30 minutes and then sonicated. The probes were incubated with Streptavidin magnetic beads for 30 minutes at room temperature to obtain probe-coated beads, followed by incubation with protein extract under gentle rotation for 2 hours at room temperature. After washing with RIP buffer, RNA-binding proteins (RBPs) were extracted for PAGE gel electrophoresis and immunoblotting. Silver-stained bands from the PAGE gel were cut out for further mass spectrometry (MS) analysis.
For the RNA immunoprecipitation (RIP) assay, the EZ-Magna RIP kit (Millipore, Billerica, MA, USA) was used to analyze the interaction between METTL13 and AGD1, according to the manufacturer’s recommendations. Briefly, cells were lysed in RIP lysis buffer and incubated with METTL13 antibody or IgG overnight at 4°C. After washing with PBS, the RNAs co-precipitated by METTL13 were extracted and analyzed using real-time PCR.
To explore the binding proteins with METTL13, a Co-IP assay was conducted. Cells were lysed with buffer (Epizyme, Shanghai, China) on ice for 30 minutes and centrifuged at 12,000 rpm for 15 minutes to remove cellular debris. The protein extract was incubated with Dynabeads Protein G (Yesean, China) with gentle shaking at 4°C for 1 hour. Subsequently, the protein-bead complexes were incubated with 5 µg of METTL13 antibody on a shaker at 4°C overnight. After washing three times with lysis buffer, the binding proteins and 10% inputs were detected by western blot.
Immunocytochemistry and immunohistochemistry
Immunocytochemistry and immunohistochemistry involved fixing PCa cells or tissue sections with 4% paraformaldehyde (PFA) and permeabilizing with 0.5% Triton-X 100 (Sigma). Blocking was performed using 5% BSA for 1 hour at room temperature, followed by overnight incubation with primary antibodies at 4°C. After two PBS washes the next day, the samples were incubated with fluorescein isothiocyanate (FITC) or rhodamine-conjugated secondary antibodies (Sigma) for 1 hour at room temperature. The nuclei were labeled with DAPI, and images were captured and analyzed using a fluorescence microscope (Nikon, Tokyo, Japan) or light microscope.
m6A dot blot assay
The dot blot assay was conducted to analyze the total m6A RNA levels in treated cells. Total RNA was extracted and denatured at 95°C for 3 minutes to disrupt secondary structures. RNA samples (400 ng, 200 ng, and 100 ng) were spotted on Amersham Hybond-N+ membranes (GE Healthcare, USA) and crosslinked by UV for 30 minutes. After blocking with 5% BSA for 30 minutes at room temperature, the membranes were incubated with m6A antibody overnight at 4°C. Following PBST washes, the membranes were incubated with HRP-conjugated secondary antibody. Images were captured and analyzed by chemiluminescence (Cell Signaling Technology) using an imaging system.
Methylated RNA immunoprecipitation (MeRIP)
Methylated RNA immunoprecipitation (MeRIP) was conducted using the MeRIP Kit (BersinBio, Guangzhou, China) following the manufacturer's instructions [40]. Briefly, 300 μg of RNA per sample, extracted from the shAGD1 and control groups, was fragmented into approximately 300 bp segments. The RNA fragments were immunoprecipitated with m6A antibody (Abcam, ab208577) or immunoglobulin G (IgG) conjugated with protein A/G magnetic beads for 2 hours at 4°C. The 10% input mRNA and m6A-enriched mRNA were used to construct RNA sequencing libraries on a Novaseq sequencer (Illumina, USA) using the PE150 strategy. MeTDiff software was employed to analyze the MeRIP-seq data [41].
RNA stability assays
Following 24 hours of plasmid transfection in PCa cells, the cells were co-cultured with 10 μg/mL actinomycin D (Yesean, China). Samples were collected at 0, 2, 4, 6, 8, and 10 hours post-treatment. Total RNA was extracted, reverse transcribed, and analyzed by real-time PCR. Three independent experiments were conducted.
Uptake of liposomal - chitosan/siAGD1 nanocomplexes
Nanocomplex uptake was examined using TEM. PC3 and DU145 cells were seeded in six-well plates and incubated overnight until reaching 60-70% confluence. Fresh medium containing 2 μM nanocomplexes was then added, and the cells were cultured for 6 hours before being switched to fresh complete medium. The cells were subsequently harvested for in vivo studies and TEM analysis. For TEM, the cells were fixed overnight at 4°C in 2.5% glutaraldehyde.
Statistical analysis
Data were analyzed using GraphPad Prism 7 (San Diego, CA) and are presented as mean ± SD. Depending on the experiment type, Student’s t-test, one-way ANOVA, and Pearson correlation coefficients were employed. A p-value of < 0.05 was considered statistically significant. All experiments were performed in triplicate.