Patients and samples collection
Craniopharyngiomas tissue samples and healthy brain tissue samples were collected at Nanchang University (Nanchang China) First Affiliated Hospital by surgery. PCPs specimens were obtained from patients who had been pathologically and clinically diagnosed with PCPs. The tumours were either kept at -80 °C until DNA/protein extraction or collected immediately for primary culture after resection. Healthy brain tissue samples were obtained from patients with conditions other than CP such as acute brain trauma or epilepsy. Written informed consent was obtained from all participants involved in the study before the clinical samples were used for research purposes. Ethical clearance was approved by the Research Ethical Committee of Nanchang University.
Primary cell culture
Fresh PCPs tissues were obtained from adult native mainly solid tumor specimens that had been directly captured after surgical removal. Only tissue including portions of tumor, as verified by instantaneous sections and microscopical inspection, was further used. After washing with phosphate-buffered saline (PBS) containing 1% penicillin/streptomycin (Life Technologies, Gibco BRL, Grand Island, USA), the specimens were dissected into pieces 2 to 3 mm3 in size and digested with 0.25% trypsin (Sigma‐Aldrich, St. Louis, MO, USA) and 1 mg/mL collagenase II (Sigma‐Aldrich, St. Louis, MO, USA) for 30 minutes at 37 °C in an incubated shaker. After centrifugation at 1000 × g at 4 °C for 10 minutes, the supernatant was discarded, and the pellet was resuspended with Dulbecco’s modified Eagle’s medium (high glucose, DMEM‐HG) with 10% fetal bovine serum (Life Technologies). Then, suspended primary PCPs cells were cultured in defined serum‐free keratinocyte medium (Invitrogen, Carlsbad, CA, USA). A constant-temperature incubator maintained at 37 °C was used to incubate cells in 5% CO2. Cell purification was carried out to establish a well‐formed primary PCPs cell line.
Cell counting kit-8 assay
4 × 103cells in suspension were seeded in a 96-well plate. Different concentrations of LY294002 were adopted to treat cells for 4 h with three replicates. Then, 10 µl CCK-8 reagent was added to the well for 4 h to form formazan. At last, the plate was read at 450–490 nm on a microplate reader.Six replicate wells were set up in each group.
Colony formation assay
A total of 5 × 102/well cells were firstly seeded in 6-well plates until most of them were attached to the bottom. Then, different concentrations of LY294002 was used for 6-well plates. After two weeks, the cells were fixed and stained. Colonies containing ≥ 20 cells were counted.
Wound healing assay
A total of 5 × 104 cells was initially seeded in each well of a 6-well plate for 24 hours. Cell layer was scratched by a pipette tip (200 µL). Subsequently, cells were then continued cultured with indicated concentrations of LY294002. Representative wound was imaged by an optical microscope. Each experiment was conducted for three times.
Cell invasion and migration assay
The Transwell cell migration and invasion assay was employed to assess cell migratory and invasive abilities. Cells were seed into 24-well Transwell Boyden chambers (8.0 µm pore size; Costar, Cambridge, MA) based on the manufacturer’s instructions. Briefly, for the Transwell cell migration assay, 5 × 104 cells were suspended in 200 µl DMEM without FBS and added to the upper chambers, then 700 µl DMEM containing 30% FBS was added into the lower chambers and incubated for 24 h at 37 °C. For the Transwell cell invasion assay, 5 × 104 cells were suspended in 200 µl DMEM without FBS and added to the upper chambers, which had been filled with 100 µl Matrigel, then 700 µl DMEM containing 30% FBS was added into the lower chambers and incubated for 48 h at 37 °C. Thereafter, non-invasive cells on the inner surfaces of the upper chambers were gently scraped off using a cotton swab. Invasive cells were fixed with 100% methanol, stained with 0.5% crystal violet solution, and washed with 1 × PBS. Following these procedures, the cells were counted and photographed using a microscope (× 100) with at least three independent experiments.
Apoptotic cell death was measured using AnnexinV-APC/7-AAD, according to the manufacturer’s protocols. Briefly, PCPs cancer cells were seeded at a densities of 5 × 104cells per well in 6-well plates, then cultured for 24 h in DMEM medium. Samples were subsequently treated with various concentrations of LY294002(0,10, 20,30,60 and 90 µM) for 24 h, after which cells were digested with 0.25% pancreatin without EDTA and washed twice with 0.001% FBS-PBS buffer. Next, 100 µL of reagent was added to micro-centrifuge tubes, after which 10 µL of cell suspension was added to each tube and incubated for 10 min at room temperature. Finally, the cells were analyzed using a BriCyte E6. Flow cytometry data was obtained from 5000 events (gated cells) per sample and the percentages of cells shown in the figures were calculated from the mean fluorescence intensity in each of the four quadrants. The coefficient of variation from the mean fluorescence was less than 10%.
Cell cycle analysis
Cell cycle assays was measured using cell cycle analysis kit (KGA512)according to the protocols. PCPs cancer cells were seeded at a densities of 5 × 104cells per well in 6-well plates, then cultured for 24 h in DMEM medium. Samples were subsequently treated with various concentrations of LY294002(0,10, 20,30,60 and 90 µM) for 24 h, after which cells were digested with 0.25% pancreatin without EDTA and washed twice with 0.001% FBS-PBS buffer. Resuspend the cells in 100 ul PBS and slowly add 700 ul pre-chilled 80% ethanol to make the final ethanol concentration 70%,at 4 ℃ fixed for 4 h, 1000 rpm, 5 min, pre-cold PBS rinse twice, add 100 ul RNase (50ug / ml), 37 °C water bath for 30 min.Next add 400 ul PI (50ug / ml) and stain at 4 °C for 30 min in the dark. Finally, the cells were analyzed using a BriCyte E6.
Primary PCPs cells were washed with PBS and then fixed in 75% ethanol for 20 minutes at room temperature. Following washing with PBS, the cells were blocked with 5% bovine serum albumin (BSA) for 1 h at 37 °C. The cells were then incubated with monoclonal anti-human pan-cytokeratin (pan‐CK, Abcam, ab215838, 1:100, Cambridge, UK), anti‐vimentin (VIM, CST, 5742T, 1:100), anti- p‐PI3K (p85) (Abcam, ab182651, 1:200), anti-PI3K (Abcam, ab191606, 1:200), anti-p‐AKT (S473) (Abcam, ab8932, 1:200), and anti-AKT (Abcam, ab8805, 1:200) overnight at 4 °C. The secondary antibody was Alexa 488–conjugated goat anti–mouse or anti–rabbit IgG (Abcam). To counterstain nuclei,4′,6‐diamidino‐2‐phenylindole (DAPI, 10 µg/mL, 32670; Sigma‐Aldrich) was used. Fluorescent images were obtained using a microscope (Carl Zeiss, Germany) and processed using Photoshop software (Adobe).
Western blotting (WB)
Total proteins were extracted from the cultured cells after treatment with or without LY294002 and PCPs tissue were prepared using RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, china). Proteins were quantified using a Pierce BCA protein Assay kit (Thermo Fisher Scientific, Inc.). Cell or tissue lysates of 30 ug protein samples were then separated by SDS-PAGE on 10—15% gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were then stained with 0.5% Ponceau S with 1% acetic acid to ensure equal loading. After blocking in PBS with Tween-20 (PBS‐T) with 5% BSA for 1 h, the PVDF membranes were incubated overnight with the following primary antibodies: monoclonal antibodies against GAPDH (Abcam, ab8245, 1:1000), p‐PI3K (p85) (Abcam, ab182651, 1:5000), PI3K (Abcam, ab191606, 1:5000), p‐AKT (S473) (Abcam, ab8932, 1:2000), and AKT (Abcam, ab8805, 1:2000). The membranes were then washed with Tris-buffered saline with Tween‐20 (TBST) (10 minutes, three times) and incubated with the secondary antibody horseradish peroxidase (HRP)-labeled goat anti-rabbit (CST, 7074, 1:4000) or anti-mouse (CST, 7076, 1:4000) for 1 h at room temperature (RT). Protein bands were visualized by enhanced chemiluminescence (ECL).
All data are presented as mean ± SEM. All experiments are performed at least in three independent times. By the means of one-way analysis of variance followed by the Duncan multiple‐comparison test using SPSS 19.0 (SPSS Inc., Chicago, IL), we calculate the statistical significance. P < 0.05, P < 0.01, or P < 0.001 is regarded as statistically significant.