Cell culture and reagents
The cell lines used in this study provided by Stem Cell Bank of Chinese Academy of Sciencesand were cultured at 37 ℃ with 5 % CO2 and 95 % air, as the instructions from the manufacturer. Briefly, cells of WPMY-1, VCaP and DU-145 were maintained in Dulbecco’s Modified Eagle’s Medium (12800017, GIBCO). Cells of 22RV1 and LNCaP were maintained in RPMI 1640 medium (31800022, GIBCO) and PC-3 were cultured in F12K medium (21127022, GIBCO). Cells were cultured in the complete mediums containing the above mediums respectively, 10 % fetal bovine serum (10099141C, GIBCO), 100 U/ml penicillin, and 100 μg/ml streptomycin.
Robustanoids A treatment on 22RV1 cells
Robustanoids A (Fig. 1a) was isolated and purified from C. canephora. 22RV1 cells were plated in 6-well plates followed by treated with 10 μM of Robustanoids A for 48 hr. In the control group, cells received 0.1% DMSO, which was tantamount to the amount in the experimental group.
Microarray analysis
After Robustanoids A treatment, cells were frozen by liquid nitrogen as soon as possible. According to Arraystar’s protocol (Rockville, MD, USA), samples were prepared and microarray hybridization were performed. Data were further remained after deletion of repeat sequences and noncoding RNAs (ncRNAs) less than 200 bp. Subsequently, hybridization was performed in Arraystar Human LncRNA Microarray V5.0, intended for comprehensive analysis of human lncRNAs and protein coding transcripts, while Agilent Scanner G2505C (Jamul, CA, USA) was used to scan data. LncRNAs which were meeting the condition of fold-changes ≥ 2 and P-values < 0.05 were considered to be significantly differentially expressed.
Patients and specimens
PCa clinical samples and the corresponding normal samples were provided by 100 PCa patients who accepted surgically resection in the Urology Department of the First Affiliated Hospital of Xinjiang Medical University in 2020 and 2021 (the ethical number: 20210301-92). The enrolled patients were pathologically confirmed and did not have androgen deprivation treatment, chemotherapy, radiotherapy, or other anticancer treatment before operation. All individuals participating in this study signed informed consents, in addition, all procedures were carried out in accordance with the ethical standards of the First Affiliated Hospital of Xinjiang Medical University.
Subcellular fractionation
Nuclei and cytoplasm were separated using the PARISTM Kit (AM1921, Thermo Fisher Scientific) based on guides from the manufacturer.
Fluorescence in situ hybridization (FISH)
The FISH test was conducted in 22RV1 and DU-145 cells according to the manufacturers’ instructions. The 5’FAM-labeled TCONS_00027385 probe (Additional file 1: S 1) in the current study was delegated to GenePharma (Shanghai, China) to design and synthesize. In brief, before permeabilization, cells were fixed in 4% paraformaldehyde (PFA) for 20 min. Subsequentially, samples were incubated overnight in a corresponding probe at 37 ℃. Finally, DAPI (D9542, Sigma-Aldrich) was used to stain the cell nuclei. Observation and photography of staining results were conducted in the fluorescence microscope (Zeiss LSM 880, Germany).
5′ and 3′ RACE
Based on the manufacturer’s instructions, a SMARTerTM RACE cDNA amplification kit (Clontech, Palo Alto, CA) was used to perform 5′ and 3′ RACE analysis methods to investigate the transcriptional start and stop sites of TCONS_00027385 (see the Additional file 1: S 2 for details).
siRNA synthesis and transfection
Specifical siRNAs which targeted TCONS_00027385 were delegated to Tsingke hz-synth department (Hangzhou, China) to synthesize, while miR-874-5p inhibitors and miR-874-5p mimics were delegated to Tsingke Biotechnology Co., Ltd. (Beijing, China) to synthesize. Transfections were performed using Lipofectamine 3000 (L3000075, Thermo Fisher Scientific) based on the manufacturer’s guide. The transfected cells were incubated in the corresponding medium for 48 or 72 hr after transfection (see the Additional file 1: S 3 & Table 1 for detail).
Colony formation test
Seeding transfected cells at 1000 cells per 10 cm plate. After 18–21 days of incubation, the cell culture medium was washed with PBS (PS102S, EpiZyme), followed by fixing in ethanol for 20 min and then staining in crystal violet for another 20 min. Finally, colonies were photographed for further count.
MTT assay
The transfected cells were seeded in 96-well plates with a density of 1 × 105 cells/well. After the treatment, cells were added and incubated with 20 μl MTT solution (5 mg/ml) (ST1537, Beyotime) for another 4 hr. Subsequentially, after carefully removing supernatants, 100 μl DMSO was added for crystal dissolution. Finally, calculating the relative value of absorbance at 490 nm measured by a microplate reader (Bio-Rad, USA) to determine the proliferation of the cells.
EdU incorporation test
Using an EdU Apollo DNA in vitro kit (C0078L, Beyotime), cell proliferation was measured by ethynyl-2-deoxyuridine incorporation test. In short, after the cells were transfected with corresponding vector and cultured, they were added and incubated with 50 μM EdU in 100 μl at 37 °C for 2 hr. Finally, cells observance were performed by a fluorescence microscopy (Zeiss LSM 880, Germany). The result was based on at least three repeats.
TUNEL assay
Cells were seeded in dishes specific for confocal microscope (801002, NEST Biotechnology), and cultured in serum-free medium for 24 hr. Subsequentially, fixing cells in 4 % PFA at room temperature for 20 min. Washing cells in 4 ℃ PBS and permeabilizing cells in 0.1 % Triton X-100 in 0.1 % sodium citrate for 10 min on ice, they were performed TUNEL staining mixture by the in situ Cell Death Detection kit, TMR red (11684817910, Roche) at 37 ℃ in the dark for 1 hr. Finally, cells were then rinsed in PBS and stained in DAPI solution for nuclei location. A LSM 880 (Zeiss, Germany) confocal microscope was used to observe fluorescence.
Apoptosis detection
The cells were collected with trypsin without EDTA and resuspended in 490 μl binding buffer. Subsequentially, samples were incubated in the dark with 5 μl Annexin V-FITC and 5 μl PI (BD 559763, BD Biosciences) for 20 mins, and then detected apoptosis stage using a FACSCalibur (BD, Biosciences, USA) within 1 hr.
Western blot detection
After cell proteins were extracted, they were separated on 10% SDS-PAGE gels, and then transferred to PVDF membranes (ISEQ00010, Millipore). Next, the membranes were blocked with 5% skimmed milk powder, and incubated with corresponding specific antibody at 4 ℃ overnight. On the next day, after incubating with appropriate secondary antibody, the expression level of protein in the samples were detected by an ECL detection system (ChemiScope 3200 Mini, China), with GAPDH as a control. In Additional file 1: S4, we have presented all the antibodies used in this experiment.
Lentivirus- induced overexpression and knockdown
To achieve TCONS_00027385 knockdown (KD) in 22RV1 and DU-145 cells, lentiviral particles within shRNAs against TCONS_00027385 or the corresponding control were obtained from Genechem (Shanghai, China). According to a previous research (20), three potential shRNAs (TCONS_00027385-KD1, TCONS_00027385-KD2, and TCONS_00027385-KD3) and a control one (TCONS_00027385-con) were designed for synthesis; the detailed sequences were presented in additional file 1: Table 2. Using polybrene transfection reagent for lentivirus transduction, and using an enhanced green fluorescent protein (EGFP) to verify and estimate the efficiency of transfection (additional file 1: Figure 1-3). Subsequentially, screening positive transformants with puromycin (MA0318, Meilun Biotechnology), the selected clones were amplified and the KD efficiency measured by qRT-PCR was analyzed. The details of experimental method for stably over-expressing cell lines can be found in additional file 1: S5 & Figure 4.
RIP test
Based on instruction for manufacturer, using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA) to perform the RIP test. Lysing the cells and incubating with Ago2 and IgG. After the cell was mixed with anti-IgG and anti-Ago2 in RIP buffer (Millipore), the RNAs in precipitation were preserved to further sequential analyze.
Luciferase reporter experiment
Using the online database, including TargetScan (http://www.targetscan.org/vert_72), ENCORI (http://starbase.sysu.edu.cn), and miRWalk (http://mirwalk.umm.uni-heidelberg.de) for prediction of binding sites. After synthesizing the wild‐type and mutant fragments related with the binding site of miR‐874-5p in the 3′‐UTR of TCONS_00027385 and ASCC2, they were inserted into the vector of pmirGLO (Tsingke Biotechnology, China). Additionally, they were mixed with miR‐874-5p mimics or inhibitors and co-transfected into 293T cells, respectively. The luciferase activity was investigated then estimated through a dual‐luciferase reporter system (Promega, Madison, WI).
Quantification of proteins by antibody array
The high-throughput protein profiles were performed using the protein array platform (AAH-APO-1-8, RayBiotech Life) based on the manufacturer’s guide. Using centrifugation to clarify cell samples (control group and sh-1 group), and before application to the arrays, a total protein concentration within the working range was obtained. Details for the experimental methods can be found in additional file 1: S6.
Zebrafish experiments
The zebrafish experiments were completed on the zebrafish platform of Zhejiang University School of Medicine (the ethical number: 2021-20515#). After fertilization, zebrafish (Danio rerio) eggs were cultured in Danieau’s solution at 28 ℃ according to standard laboratory conditions. At 48 hr after fertilization, the membranes out of the embryos were carefully removed with forceps, and the embryos were anesthetized in 0.04 mg/ml tricaine. The microinjection was performed to the embryos after transferred to a modified agarose gel. Using a Pneumatic Picopump and a manipulator (WPI) to inject approximately 400 DU-145 cells on the ventral end of the Cuvier Duct, where which entered embryo’s heart. The injections were repeated in at least 15 embryos in each group. The survival rate less than 85 % in the control group was regarded to the demarcation line for abandonment. After implantation, embryos were cultured at 33 ℃ (21). Every other day, the growth of tumor was observed through a SMZ18 fluorescence microscope (Nikon, Japan) while green pixels data were quantified by NIS-Elements imaging software (D 4.50.00).
Tumor xenograft model
After 22RV1 cells (1×107 cells) with or without TCONS_00027385-KD1 (sh-1) were suspended in 200 μl PBS, they were injected subcutaneously into nude mice of 4-6 weeks old in each side. 30 days after injection, the mice were sacrificed for measurements of tumors in the terms of maximum (L) and minimum (W) length and weight. This animal experiment had been approved by the Animal Care and Use Committee of Zhejiang University (the ethical number: 2021-20515#).
Statistical analysis
Analyzing the data of at least three in-dependent tests with the GraphPad Prism 7.04 software (La Jolla, USA) and expressing them in the form of mean ± S.D. The student’s t-test was performed to compare the differences between two groups, while the survival rate was calculated through Kaplan–Meier survival analysis. In addition, analysis of Cox proportional hazard model multivariate was conducted to measure the significance of TCONS_00027385 expression and clinicopathological characteristics on overall survival. Statistically significant was defined as P-value < 0.05.