Patients and samples
AIPC samples were obtained from 45 patients who provided informed consent, which was in accordance with the ethical standards of the Tongren Hospital (Shanghai, China) Review Board. All samples were reviewed by a pathologist and were confirmed as AIPC based on a histopathological evaluation. No local or systemic treatments were administered in these patients before surgery.
The AIPC cell lines PC3 and DU145 were obtained from the American Type Culture Collection (Manassas, VA, USA). The normal prostatic cell line RWPE was purchased from Jennio Biotech Co(Guangzhou, China). PC3 and DU145 cells were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Gibco). RWPE cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS. All cells were incubated at 37 °C in a 5% CO2 humidified incubator.
The LEF1-AS1 pcDNA3.3 vector and empty vector (pcDNA3.3) were based on the expression vector pcDNA3.3 (Invitrogen, USA). Small interfering RNA for LEF1-AS1, C-myb, FZD2 or CD44 (siLEF1-AS1, siC-myb) and scramble siRNA (si NC) were purchased or synthetized from RiboBio (Guangzhou, China). The cells were seeded into 6-well plates, and transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
RNA isolation and RT-PCR analyses
Total RNA was extracted from AIPC samples and cells using TRIzol reagent (Invitrogen), and cDNA was synthesised using the QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. qRT-PCR was then performed based on the instructions for SYBR Premix Ex Taq (Takara, Tokyo, Japan), and the expression levels were normalized to the level of GAPDH. Each experiment was repeated at least three times.
Chromatin immunoprecipitation (ChIP)
To verify the potential transcription factors binding to the LEF1-AS1 promoter, a ChIP assay was performed using the EZ-Magna ChIP kit (Millipore, Shanghai, China) according to the manufacturer’s protocol. In brief, cells were fixed with 4% paraformaldehyde and incubated with glycine for 10 min to generate DNA–protein cross-links. Then, the cells were lysed with Cell Lysis Buffer and Nuclear Lysis Buffer and sonicated to generate chromatin fragments of 400–800 bp. The chromatin fragments were immunoprecipitated with an anti-C-myb antibody (Cell Signaling Technology, Danvers, MA, USA) or control IgG. The resulting DNA was purified for PCR analyses.
RNA binding protein immunoprecipitation (RIP) assay
RIP was performed according to the protocol for the EZ-Magna RIP kit (EMD Millipore, Billerica, MA, USA). Briefly, DU145 cells grown to 80–90% confluency were lysed in complete RIP lysis buffer, and 90% of 100 µL of whole cell extract was incubated with RIP buffer containing magnetic beads conjugated with 5 µg human anti-C-myb antibody (Abcam) or immunoglobulin G (IgG) control. Incubation with proteinase K with shaking was performed to digest the protein, and RNA was isolated by immunoprecipitation. Finally, the immunoprecipitated RNA was purified and analysed by qRT-PCR.
Cells (5 × 106) were lysed for 20 min with lysis buffer (Beyotime Biotechnology) containing protease inhibitors (Roche, Indianapolis, IN, USA). Equal amounts of samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% (wt/vol) skimmed milk in TBS plus Tween 20 at 4 °C overnight before probing with antibodies. Membrane proteins were detected by an HRP-conjugated secondary antibody (1:1000, Santa Cruz Biotech, Santa Cruz, CA).
Colony formation assay
AIPC cells were collected, and 1000 cells were seeded in each well of a 6-well plate. After cell culture for 15 days, PC3 or DU145 cells were washed with PBS and fixed in methanol for 20 min. After washing three times with PBS, colonies were stained with 0.1% crystal violet for 20 min. Colonies were captured using a light microscope (Olympus, Tokyo, Japan). Clusters containing ≥ 30 cells were counted as a single colony.
The cells were cultured in 96-well plates at a density of 2 × 103 cells/well. Then, the EDU (5′-ethynyl-2′-deoxyuridine) incorporation assay was performed to evaluate cell proliferation using a KeyFluor488 Click-iT EDU kit (KeyGENBioTECH, Nanjing, China) according to the manufacturer's instructions. The percentage of Edu-positive cells was calculated after fluorescence microscopy analysis.
Cell migration and invasion assays
The cells were harvested, resuspended in serum-free media and placed into the upper chamber of a Transwell membrane filter (Corning, NY, USA) for the migration assays or in the upper chamber of a Transwell membrane filter coated with Matrigel (Corning) for the invasion assays. After 24 h of incubation, cells on the upper side were removed with a cotton swab. Evaluation of invasive capacity was performed by counting invading cells under a microscope (40 × 10). Five random fields of view were analysed for each chamber.
AIPC and adjacent tissues were fixed in 4% paraformaldehyde for paraffin embedding. Tissue specimens were cut into slices, and tissue slices were deparaffinized, rehydrated, and immersed in 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. Cancer tissues were immunostained for FZD2 and CD44 (Abcam, San Francisco, CA, USA; 1:100 dilution). The signal was amplified and visualized with diaminobenzidine chromogen, followed by counterstaining with haematoxylin.
Tumourigenicity assays in nude mice
We used male nude mice (6 weeks of age) obtained from the Animal Facility of Shanghai Jiao Tong University School of Medicine; the mice were fed sterilized food and water. All of the animal experiments were approved by the responsible governmental animal ethics committee and complied with the ARRIVE guidelines. Thirty-day-old male nude mice were subcutaneously injected with 1 × 106 AIPC cells in 200 µL serum-free medium into the right flank. Once palpable tumours were observed (after approximately 4 weeks), the mice were sacrificed. The tumours were isolated and weighed.
The results are presented as the mean ± SD from three independent experiments performed in triplicate. The P values were calculated by using Student t test or one-way ANOVA. A P value of < 0.05 was considered to indicate a statistically significant result. Statistical analyses were performed using GraphPad Prism 6.0 statistical software (GraphPad Software Inc., La Jolla, California).