Patient tissue specimens and cell lines
A total of 60 sets of PCa tissue specimens and their corresponding normal prostate tissues were obtained from patients who underwent prostatectomy for PCa at the Department of Urology of the Union Hospital affiliated with Tongji Medical College between 2015 and 2019. Before sample collection, approval was obtained from the Institutional Review Board of the Tongji Medical College of the Huazhong University of Science and Technology. All specimens were classified according to the 2004 World Health Organization Consensus Classification and Staging System for prostate neoplasms. The anoikis-resistance model and cell culture were established using human androgen-independent PCa cell lines, PC-3 and DU145, which were obtained from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). Androgen-dependent C4–2 and LNCaP cell lines were donated by Prof. Xiaoping Zhang and Professor Jun Zhao (Union Hospital, Wuhan, China). The prostatic epithelial cell line RWPE-1 and androgen-dependent 22RV1 were obtained from the China Center for Type Culture Collection, CCTCC (Wuhan, China). The cells were maintained in RPMI 1640 medium (Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (Biologic Industries, Kibbutz Beit Haemek, Israel) and 1% penicillin/streptomycin (Beyotime Institute of Biotechnology, Nanjing, China) at 37 °C in 5% CO2 and 95% humidity. The anoikis-resistance model was established by continuously culturing corresponding parental (P) cells in ultra-low-attachment 6-well plates (Corning, NY, USA) for 7 days, after which they were transferred to standard plates to allow adherence for 24 h. Re-adherent cells were deemed anoikis-resistance [12, 23, 24].
RNA extraction, RNase R treatment, and PCR assays
The total RNA was isolated from the tissue and cell lines using a RNeasy Mini Kit (QIAGEN, Germany) according to the instructions of the manufacturer. RNase R treatment occurred at 37 °C with 3 U/mg of RNase R (Epicenter, WI, USA) for 15 min. Complementary DNA was synthesized using random primers and a PrimeScript RT Master Mix reverse transcription kit (Takara, Dalian, China) or a commercial miRNA reverse transcription PCR kit (Ribo-Bio, Guangzhou, China). Genomic DNA (gDNA) was isolated using a QIAamp DNA Mini Kit (QIAGEN, Germany), while quantitative real-time PCR (qRT-PCR) analysis was performed using an SYBR Premix Ex TaqTM kit (Takara). The differences between the circRNA and miRNA were normalized to GAPDH or U6 levels. All data were analyzed using the StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). Bulge-loop miRNA qPCR primers were obtained from RiboBio (Guangzhou, China). The primer details are listed in Supplementary Table 1.
RNA-fluorescence in situ hybridization (FISH)
Cy3-labeled circ_0004585 and Dig-labeled locked nucleic acid miR-1248 probes were purchased from RiboBio (Guangzhou, China). The images were obtained using a fluorescent in situ hybridization kit (RiboBio) according to the instructions of the manufacturer. All data were analyzed using a Nikon A1Si laser scanning confocal microscope (Nikon Instruments Inc., Japan) and ModFit LT software.
Pull-down assay with a biotinylated-A probe
The biotinylated-circ_0004585 probe was synthesized by RiboBio (Guangzhou, China). The sequence of the probe was just complemented to the back-spliced junction of circ_0004585 (listed in Supplementary Table 4). The pull-down assay with biotinylated miRNA was performed as previously described [25, 26] The RNA complexes combined on the beads were finally extracted using an RNeasy Mini Kit (QIAGEN, China) for further assessment. The pull-down assay with biotinylated miRNA miR-1248 mimics or mutants was synthesized by RiboBio (Guangzhou, China), while the bound RNAs were purified using a RNeasy Mini Kit (QIAGEN) for further analysis.
Apoptosis detection
Non-transfected cell apoptosis was detected using FITC V Apoptosis and PE Annexin V Apoptosis Detection Kits (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, cells (5 × 105) were collected and incubated with FITC/propidium iodide (PI) for 15 min in the dark at room temperature, while the apoptosis index was determined using a flow cytometer (Beckman Coulter, Indianapolis, IN, USA). For the detachment-induced apoptosis assay, cells were incubated in ultra-low-attachment plates for 24 h before detection. PC-3 and DU145 cells were treated with 200 nM rapamycin (Rapa), or 10 mM 3-methyladenine (3-MA) (Selleck Chemicals, Houston, TX, USA) and 10 μM Chloroquine (CQ) (Selleck Chemicals, Houston, TX, USA) for 24 h, respectively, while isometric double-distilled water or DMSO was used as negative controls (NC). The reagents and drugs used in the study are detailed in Supplementary Table 2.
Wound healing assay
PC-3 and DU145 cells were cultured in 6-well plates and scraped with the fine end of a 200 μl pipette tip (time 0 h). The cell migration was photographed 0 h and 24 h after injury using ten high-power fields. Remodeling was measured as the diminishing distance across the induced injury, normalized to the 0 h control, and expressed as relative migration.
Transwell migration and Matrigel invasion assays
The cell migration was evaluated using a 24-well Transwell plate with 8.0-mm pore polycarbonate membrane inserts (Corning). Homogeneous single-cell suspensions (200 μl; 1 × 105 cells/well) in serum-free mediums were added to the upper chambers, while 500 μl complete mediums were added to the lower chambers. After incubation for 24 h at 37 °C in a CO2 incubator, the migrated or invaded cells were fixed with ice-cold methanol and stained with 0.1% crystal violet for 15 min at room temperature. The migrated cells were counted in three randomly chosen fields using an inverted phase-contrast microscope (Olympus, Tokyo, Japan) at 100× magnification.
Cell Counting Kit-8 (CCK-8) assay
The cell viability was determined using a CCK-8 assay (Supplementary Table 2). The PCa cells were starved in a medium containing 0.1% fetal bovine serum for 24 h. Next, approximately 2 × 103 cells were evenly seeded into 96-well plates in triplicate. Then, CCK-8 solution (10 μl) was added to each well and incubated at 37 °C for 2.5 h. The OD value was measured at 450 nm after 1 d, 2 d, 3 d, 4 d, and 5 d, respectively, using a SpectraMax M5 microplate reader (MD, USA). The cell doubling times were calculated using GraphPad Prism 7.0 software (La Jolla, USA), while the OD values were used to perform the statistical analysis.
Immunofluorescence staining assay
The mCherry-GFP co-labeled LC3BII/I adenovirus (Beyotime) was transferred to PCa cells in different treatment groups for 24 h [27]. Autophagy flux were detected in 2D culture, and a total of more than 20 cells under each condition were counted for quantification of autophagy and the images were amplified by 200 times. and the images were magnified 600 times. The nuclei were stained with DAPI, and the data were analyzed using a Nikon A1Si laser scanning confocal microscope (Nikon Instruments Inc., Japan).
Transmission electron microscopy (TEM)
The cells were collected and fixed with 2.5% glutaraldehyde (Sigma, G7526) at 4 °C for 2 h. TEM of the autophagic and autolysosome ultrastructures in the cells was performed as described in a previous study [28]. The images were acquired using an 11-megapixel CCD camera (Olympus, Japan).
Immunohistochemistry analysis
The primary antibody used to detect TM9SF4 was purchased from the Proteintech Group (Chicago, USA). The immunostaining images were captured using an Olympus FSX100 microscope (Olympus, Japan). The immunohistochemical expression level of TM9SF4 was evaluated by H-score, which was jointly completed by two pathologists in our hospital. The protein expression levels were analyzed by calculating the integrated optical density as described [29].
Luciferase reporter assays
The TM9SF4 3′-UTR or promoter reporters were transiently transfected along with the Renilla control plasmid, while the PCa cells were co-transfected with miR-1248 mimics. The luciferase activities were measured for 24 h using a dual-luciferase reporter assay detection kit (Promega, WI, USA) as previously described [30].
Plasmid construction and stable transfection
The human circ_0004585 and TM9SF4 3′-UTR cDNA was synthesized by TSINGKE (Wuhan, China). Circ_0004585 was cloned into a pCD-ciR vector (Geenseed Biotech Co, Guangzhou, China) containing front and rear circular frames. The TM9SF4 3′-UTR was cloned into a pMIR-REPORT vector. The T1 and T2 TM9SF4 3′-UTR plasmids and mutant luciferase reporters were synthesized using a Trelief™ SoSoo Cloning Kit (TSINGKE, Beijing, China). The TM9SF4 promoter-luciferase reporter vector was constructed and used as described in previous studies [31]. The miR-1248 mimics and the control were purchased from RiboBio (Guangzhou, China), while the siRNA aimed at circ_0004585 was synthesized by Gene-Pharma (Shanghai, China). The shRNA targeting TM9SF4 was designed and synthesized by Genechem (Shanghai, China). The targeted sequence was in Supplementary Table 5. while Lipofectamine 2000 (Life Technologies, USA) was used for plasmid transfection according to the instructions of the manufacturer. The transfected PCa cells were screened for four to six weeks using G418 (Life Technologies, USA).
Western blots
The cell lysates were prepared with RIPA buffer (Thermo Scientific), the concentration of which was determined using a bicinchoninic acid protein assay kit (Pierce, Thermo Scientific). The immunoreactive bands were determined with an Immobilon ECL substrate kit (Millipore, Merck KGaA, Germany), while the images were acquired using a BioSpectrum 600 Imaging System (UVP, CA, USA). Detailed information regarding the primary antibodies used in this study is listed in Supplementary Table 3.
Tumor xenografts
Four- to five-week-old male BALB/c nude mice were selected for the tumor xenograft experiments. PC-3 cells, labeled with Cy3 and transfected with overexpressed or knockdown circ_0004585 plasmids or control vectors, were subcutaneously injected into the tail veins of the mice (3 × 106, 200 μl), who were sacrificed after six to seven weeks. No blinding occurred during the animal experiments. Xenograft images of the mice were obtained using the In-Vivo FX PRO (BRUKER Corporation, USA). All procedures were approved by the Animal Care Committee of Tongji Medical College.
Statistical analysis
Statistical analysis of the data was performed using GraphPad Prism 7.0 software (La Jolla, USA), and the results were expresses as mean ± standard error of the mean (SEM). The two groups were compared via a two-tailed Student’s t-test. One-way analysis of variance was performed to evaluate the group differences, while P < 0.05 was considered statistically significant.