The LNCaP, 22RV1 and PC3 cell lines were generously provided by the Steam Cell Bank, Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in RPMI 1640 medium (L210KJ; BasalMedia, Shanghai, China) supplemented with 10% fetal bovine serum (S660JJ; BasalMedia), 1% penicillin/streptomycin (15070063; Gibco, Grand Island, NY, USA), and 1% HEPES (15630080; Gibco). The cells were grown at 37℃ in a humidified incubator under 5% CO2.
Plasmids and lentiviral infection
The following short hairpin RNA (shRNA) expression sequences were used: DDX52, 5′-GCCAATCCAAATGCAAGCCAT-3′; c-Myc, 5′-CCCAAGGTAGTTATCCTTAAA-3′; and control, 5′-GCTCCGTGAACGGCCACGAGT-3′. These sequences were cloned into the PLKO.1 vector (10879; Addgene, Watertown, MA, USA). The overexpressed DDX52 plasmids were cloned into the pLVX-IRES-ZsGreen1 vector (Takara Bio, Shiga, Japan) via DNA assembly (#E5520; NEB, Rowley, MA, USA).
The psPAX2 plasmid (12260; Addgene) and pCMV-VSVG plasmid (8454; Addgene) in 293 T cells were transfected into HEK293T cells with PEI 25K (23966-1; Polysciences, Warring, PA, USA) following the manufacturer’s instructions. The culture medium was collected after 48 and 72 h, and the supernatant was centrifuged at 1000 rpm for 10 min to obtain the supernatant products containing the lentivirus to transduce into the indicated cells. Puromycin (5 µg/ml) (Sigma–Aldrich, St. Louis, MO, USA) was used to isolate the stable transformants in PC3 and 22RV1 cells.
Cell growth and colony formation assay
The Cell Counting Kit-8 was used to measure cell growth according to the manufacturer’s instructions (CK04-1000T; Dojindo Technologies Inc., Rockville, MD, USA). The 22RV1 (1500 cells/well) and PC3 cells (1500 cells/well) were plated in 96-well plates and assessed at the indicated times after seeding.
The 22RV1 (700 cells/well) and PC3 cells (700 cells/well) were also seeded in six-well plates in complete medium for 6–14 days depending on the size of the colony. Then, the cells were fixed in methanol for 10 min and stained with 0.1% crystal violet for 1 h.
Approximately 1 × 106 lentivirus-infected 22RV1 cells in Matrigel (1:1 [v/v], 356234; Corning, Inc., Corning, NY, USA) were injected into 6-week-old male athymic nude mice (n = 7) under anesthesia by intraperitoneal injection of 2% Tribromoethyl alcohol (125 ~ 400 mg/kg; HY-B1372; MedChem Express, Shanghai, China). A tumor-free status was defined as a xenograft that did not touch the flank of the male nude mice. All mice were sacrificed 6 weeks later, and the xenografts were dissected and weighed. The euthanasia of mice was performed by inhaled CO2 for the concentration of 60% for 10 min, and then to rise to 100% for 30 min. All animal experiments were approved by the Experimental Animal Ethics Committee of the Department of Laboratory Animal Science, Fudan University.
Western blotting and IHC
The cells were washed twice in phosphate-buffered saline and solubilized in lysis buffer. Approximately 40 µg of protein sample was separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Immobilon IPVH304F0; Sigma–Aldrich). The membrane was blocked in 5% bovine serum albumin in Tris-buffered saline and Tween and incubated with primary and secondary antibodies.
The tissues were fixed in 10% buffered formalin for 24 h and embedded in paraffin. The paraffin-embedded tissues were sectioned and placed on charged glass slides, followed by hematoxylin and eosin or IHC staining using an IHC staining kit (G1215-200 T; Servicebio, Wuhan, China) following the manufacturer’s instructions. IHC scores were calculated using the formula: IHC score = intensity score × percentage score. The intensity score was estimated according to staining intensity (0: negative, 1: weak, 2: moderate, and 3: strong); the percentage score was determined according to the percentage of stained cells (0: 0, 1: 1–25, 2: 26–50, 3: 51–75, and 4: 76–100%). Antibodies against the following were used: DDX52 (NBP2-33776; Novus Biological, Centennial, CO, USA; western blot [WB]: 1:2,000, IHC: 1:500), c-Myc (ab32072; Abcam Cambridge, MA, USA; WB: 1:1,000, IHC: 1:100), Ki67 (sc-15,402; Santa Cruz Biotechnology, Santa Cruz, CA, USA; IHC: 1:200), AR (sc-816; Santa Cruz Biotechnology, Santa Cruz, CA, USA; WB: 1:1000), GAPDH (sc-365,062; Santa Cruz Biotechnology, Santa Cruz, CA, USA; IHC: 1:200), and vinculin (66305–1-Ig; Proteintech, Rosemont, IL, USA; WB: 1:1,000).
RNA sequencing and analysis
Total RNA extracted from the indicated groups of 22RV1 and PC3 cells was subjected to RNA sequencing (RNA-seq) performed by Majorbio Biopharm Technology (Shanghai, China). The sequencing reads were analyzed and mapped using the free online Majorbio Cloud Platform (www.majorbio.com) to acquire the expression profiles. The sequencing data were submitted to the Gene Expression Omnibus (GEO; GSE171797).
The expression profiles published by Yu et al. (GSE6919) , Grasso et al. (GSE35988) , and Penney et al. (GSE62872)  were downloaded from the GEO. Expression profiles from Abida et al. (SU2C/PCF Dream Team) , the Cancer Genome Atlas, and Kumar et al. (Fred Hutchinson Cancer Research Center)  were downloaded from cBioPortal (https://www.cbioportal.org/) [14, 15]. Gene set enrichment analysis (GSEA)  was performed using software provided by the Broad Institute (http://www.broadinstitute.org/gsea/index.jsp) following the website’s guidelines. We used the curated gene sets (hallmark gene sets) within MSigDB.
All statistical analyses were performed using GraphPad Prism software (version 9; GraphPad Software Inc., La Jolla, CA, USA). The statistical analysis methods are described in the figures or figure legends. A p-value < 0.05 was considered to indicate significance.