Ethic statement
This study was approved by Ethics Committee of Zhujiang Hospital, Southern Medical University. The informed consents were signed by all the patients. According to the ethical and legal standards, every specimen was made and handled anonymously. All animal experiments in this study were carried out following the guidelines of the Institute for Laboratory Animal Research at Southern Medical University, Guangzhou, P. R. China.
Patient samples
Prostate cancer tissues and benign prostate hyperplasia tissues were respectively collected from 10 patients of Cancer Center of Guangzhou Medical University (Guangzhou, China) between 2018 and 2020. All the clinical and pathological information were summarized in Supplementary Table 2 and Table 3. Fresh tissues were viewed and approved by two pathologists, frozen immediately in liquid nitrogen, and stored at − 80 °C.
Cell culture
All the human prostate cancer cell lines including PC3, DU145, LNCaP and benign prostate hyperplasia cell line BPH-1, and human embryonic kidney cells (293T) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). According to the instructions, PC3 was cultured in F12K medium (Procell), DU145 was cultivated in DMEM medium (Gibco), while 293T, LNCaP and BPH-1 were maintained in RPMI-1640 medium (Gibco). All media listed above were supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% Penicillin-Streptomycin (Gibco). All cell lines were maintained at 37 °C with 5% CO2. For the autophagy induction experiments, all stable or transient transfected cell lines were maintained in Earle’s balanced salt solution (EBSS) in the presence of 10μM Bafilomycin A1 (BAF) and/or 50nM Chloroquine (CQ) for 8 h. Dimethyl sulfoxide (DMSO) was used as negative control.
Transfection
The has_miR-520h mimics, pcDNA3.1-EGR1 and small interfering RNAs (siRNAs) targeting circCSPP1 or HnRNP-L were synthesized and transfected into cell lines using siRNA-Mate or GP-transfect-Mate (Genepharm) following the manufacturer’s instruction. Lentivirus vectors encoding HnRNP-L, sh-/circCSPP1, sensGFP-stubRFP-LC3 and AGO2 were constructed and transfected into prostate cancer cell lines with Hitrans-GP (GeneChem). All the lentiviral transfected cells were treated with 1 μg/mL puromycin for 7 days to generate the stable cell lines. siRNA and miR-520 mimics sequence details are shown in Supplementary Table 1.
RNA extraction and real-time PCR
Total RNA was isolated from cells by using TRizol reagent (Takara, Tokyo, Japan) according to the instructions of manufacturer. Nuclear and cytoplasmic RNA fractions were separated from 102-107 cell pellets with the PARIS™ Kit (Ambion, Lifetechnologies) according to the manufacturer’s instructions. RNA sample was treated with RNase R (Geneseed) at 37°C for 30 min to obtain purified circRNA. For circRNA and mRNA, cDNA was reverse-transcribed by using HiScript II Q RT SuperMix for qPCR (R223-01, Vazyme). For miRNA, cDNA was synthesized by using PrimeScript™ RT reagent Kit with gDNA Eraser (RR0471, Takara) with Bulge-Loop™ miRNA RT Primer (R10031.7, RIBOBIO). RT-qPCR was carried out using the SYBR Green Realtime PCR Master Mix (QPK-201, TOYOBO) with the CFX connect qPCR Detection System (Bio-Rad). β-actin was used as the endogenous control for mRNA and circRNA while U6 for microRNAs to calculate the relative fold changes for transcript abundance. Primers sequence details are shown in Supplementary Table 1.
Nucleic acid electrophoresis
The cDNA was augmented by RT-qPCR and gDNA was amplified using 2×Taq PCR MasterMix by T100™ Thermal Cycler (Bio-Rad). Then all type of PCR products were separated by 2% agarose gel electrophoresis with TAE running buffer. The electrophoresis was running at 110V for 40 min. Finally, the gels were irradiated by Ultraviolet rays and the targeted gene bands were measured by comparing with the DNA marker DL2000 (3427A, Takara).
Western blot analysis
For detecting relative autophagy markers LC3 and P62, cancer cells were treated with EBSS containing 10μM Bafilomycin A1 (BAF) or 50nM Chloroquine (CQ) for 8 h before lysed by RIPA buffer with PMSF on ice for 15min. Then the cell lysis was mixed with 5× protein loading buffer and was subsequently denaturalized at 100℃ for 10 minutes. Total protein denaturants were separated by SDS-PAGE, transferred onto PVDF membranes (Millipore) and blocked with 5% skim milk in TBST for 1h. The membranes were incubated with primary antibodies against β-actin (BA2305, Boster), LC3A (NB100-2331, Novus), SQSTM1/P62 (sc-28359, Santa Gruz), HnRNP-L (ab6106, abcam), EGR1 (#4154 CST) at 4℃ overnight. Then, all the membranes were immersed in horseradish peroxidase-linked secondary antibodies against rabbit or mouse IgG. The bands were visualized using chemiluminescence imaging system (CLiNX ChemiScope Touch, Shanghai) and quantified by imageJ software.
RNA Immunoprecipitation assay
2×107DU145 cells were collected and lysed by ice-cold polysome lysis buffer with protease inhibitor and RNase inhibitor from the RNA Immunoprecipitation Kit (Bes5101, BersinBio). Major part (90%) of the cell lysis were incubated with anti-HnRNPL or AGO2 (IP group) and non-specific IgG (IgG group) respectively on vertical mixer at 4℃ for 16h, while the other was kept as an input group. Subsequently, two groups were mixed with protein A/G beads by vortex at 4℃ for 1h, followed by recovery of beads and RNA elution from the mixture. The RNA samples were quantified, reversely transcribed to cDNA and RT-qPCR analysis among IP, IgG and Input group.
Fluorescence in situ hybridization (FISH)
1 ×104cells were embedded to cover slide in a 48-well plate and cultured overnight. PCa cells were fixed by 4% paraformaldehyde for 15 min at room temperature after washed with 1X phosphate-buffered saline (PBS) for 5 min x 2 and then penetrated with 0.1% Triton X-100. Subsequently, cells were washed with 1X PBS for 5 min x 2 and treated with 2× SSC for 30 min at 37 °C. The probes (PA20190412001, RiboBio) targeting circCSPP1/EGR1/has_miR-520h were pre-mixed with hybridization buffer and denatured at 73 °C for 5 min. After hybridization, the slides were washed with 0.1% Tween 20 for 5 min at 42°C, and then washed with 2 × SSC at 42°C for 5 min × 2. DAPI was re-dyed at dark room temperature for 20 min and washed with 1X PBS, 5 min x 2. Treatment of antifade reagent and the cell slides were adhered to the slide (face up) with neutral gum before the observation under fluorescence microscope.
Luciferase Reporter Assay
Dual luciferase reporter vector pmirGLO (Promega) was used for the luciferase assays. circCSPP1/EGR-1 wild-type (circCSPP1/EGR-1 WT) and mutant (circCSPP1/EGR-1 MUT) reporter vectors were constructed and inserted into the pmirGLO. 5 ×105cells were seeded into a 12-well plate and cultured for 24h at 37℃ with 5% CO2. Subsequently, 1.6μg reporter plasmids (circCSPP1/EGR-1 WT, circCSPP1/EGR1 MUT) together with 20μM has_miR-520h mimics or negative control were transfected into DU145/PC3 cells. Then the transfected cells were transferred to the incubator and cultured for another 48h. Finally, the Dual-Luciferase Reporter System Kit (Promega) was used to detect the luciferase activity with Tecan M1000 microplate reader.
Electron microscopy
Adherent cells estimated at 1 ×106 were treated with 0.25% trypsin for only 30 seconds to keep cell membrane intact. Cell suspension was centrifuged at 800 rpm for 5min following by supernatant removal. Each sample was fixed with 2% glutaraldehyde at 4℃ for over 15 min and washed with PBS three times for 10min each. Samples were post-fixed with 1% OsO4 followed by an ascending gradient dehydration step of ethanol and infiltration with propylene oxide. After ultrathin sectioning and staining with 3% lead citrate- uranyl acetate, samples were observed under an electron microscope (HT-7800, Hitachi High-tech).
Examination of autophagy flux
PC3 and DU145 cells were cultured and transfected with lentivirus carrying sensGFP-stubRFP-LC3 at 37 °C for 48 h. Next, the transfected cells were treated with EBSS containing 50μM BAF for 8 h. Cells were fixed with 4% paraformaldehyde for 30 min. Finally, the autophagy flux was analyzed using a confocal fluorescence microscopy (Leica, Germany). In merged images, yellow spots represent autophagosomes while red spots represent autolysosomes.
Cell proliferation and Colony formation assays
Cell proliferation was determined using CCK-8 assays (MA0218-5, Meilunbio). The transfected cells were seeded into 96-well plates at a density of 3000 cells per well. 100μl complete medium containing 10μl CCK-8 reagent was added into each well at 0, 24, 48, 72, 96h after seeding. All plates were scanned using a microplate reader (Bio-Rad) in another 2h incubation. The absorbance at 450nm was measured and analyzed. PCa cells were plated at an initial density of 500 cells per well in a 6-wells plate and cultured at 37°C with 5% CO2 for 14 days. Then, the colonies were fixed for 20 min with 4% paraformaldehyde and stained for 15 min with crystal violet. After discarding the staining solution, the plates were air-dried at room temperature and then observed under the light microscope.
Migration and invasion assay
Wound healing assays were carried out to evaluate the migration ability of PCa cells. Transfected cells were seeded in 6-well plates at a density of 1 ×106 cells/well and grown to 90-100% in 10% FBS medium. Then linear wounds were scratched with a sterile 200μL plastic pipette tip in each well and PBS was used to remove the detached cells. Cells were cultured in FBS free medium to inhibit cell proliferation. Images of the scratched area was captured at indicated times (0 and 24h) using a Leica light microscope. For invasion assay, the transwell chamber was precoated with Matrigel (), and 6×104 cells were seeded to the upper chamber. Subsequently, 500 μL of DMEM medium containing 10% FBS was added to the lower chamber. The cells on the top surface were removed with a cotton ball after incubation for 24 h, and the cells that invaded to the lower membrane surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. The invaded cells were then photographed and counted under an inverted microscope.
Animal experiments
The 4-week old C57BL/6 nude mice (male) were obtained from Guangdong Experimental Animal center (Guangzhou, China). 6 male C57BL/6 mice were subcutaneously injected with 3 × 10^6 stably-transfected DU145 cells (empty vector or circCSPP1-overexpression) in both back sides. The growth of implanted PCa tumors was monitored by measuring their volumes every 3 days. Finally, the mice were sacrificed and their xenografts were measured and photographed.
Statistical analysis
All data were shown as mean ± SD processed by GraphPad Prism 7.0 (La Jolla, USA). Student’s t-test analysis was used to evaluate the normalized data. Pearson correlation assay was used to analyze expression correlation (circCSPP1, miR-520h and EGR1). Kaplan-Meier method was used to estimate the Overall survival (OS) and Biochemical recurrence (BCR) curve. All statistical tests were two-sided and considered statistically significant when p values are less than 0.05.