Patients and sample collection
This study was carried out after approval by the Ethics Committee of West China Hospital, Sichuan University (Sichuan China), and written informed consent was obtained from all participants. A total of ten tumor tissue samples were collected from patients with craniopharyngioma at West China Hospital, Sichuan University (Sichuan China). The inclusion criteria were as follows:
(1) Primary surgical removal in the neurosurgical department of West China Hospital, Sichuan University;
(2) No preoperative or postoperative radiotherapy or chemotherapy;
(3) Histological diagnosis of craniopharyngioma.
Cell culture
Primary cell culture was performed as described previously[6]. Briefly, the tumor sample was washed with phosphate‐buffered saline (PBS) containing 1% penicillin/streptomycin (Life Technologies, Gibco BRL, Grand Island, USA), dissected into pieces and digested with 0.25% trypsin (Sigma‐Aldrich, St Louis, MI, USA) and 1 mg/mL collagenase II (Sigma‐ Aldrich, St Louis, MI, USA) for 30 minutes at 37°C in an incubated shaker. The cells were cultured with DMEM (Life Technologies, Gibco BRL, Grand Island, USA) containing 10% FCS (Life Technologies, Gibco BRL, Grand Island, USA) in an atmosphere at 37°C with 5% CO2.
RNA isolation and quantitative reverse transcription PCR
Total RNA was extracted from samples using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. The complementary DNAs (cDNAs) were synthesized using the Reverse Transcription System Bestar qPCR RT Kit according to the manufacturer’s instructions with the ABI 7500 Real‐Time PCR System (Applied Biosystems, Lincoln Center Drive Foster City, CA 94404, USA) and reverse-transcribed with M-MLV reverse transcriptase (Promega, Madison, Wisconsin, USA). U6 and GAPDH were used as internal controls. The primers were as follows:
miR-200b:
forward, ACACTCCAGCTGGGTAATACTGCCTGGTAA,
loop primer, CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG TCATCATT;
miR-200a:
forward, ACACTCCAGCTGGG TAACACTGTCTGGTAA,
loop primer, CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG ACATCGTT;
miR-200c:
forward, ACACTCCAGCTGGG TAATACTGCCGGGTAAT,
reverse, CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCCATCAT;
miR-429:
forward, ACACTCCAGCTGGG TAATACTGTCTGGTAA,
loop primer, CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACGGTTTT;
miR-141:
forward, ACACTCCAGCTGGGTAACACTGTCTGGTAA,
loop primer, CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCATCTTT;
ZEB1:
forward, ATGTGGCTAGTTTGTCCTC,
reverse, AGCAAGATTTCCTCCAGGTC;
ZEB2:
forward, TCTGCGACATAAATACGA,
reverse, GAGTGAAGCCTTGAGTGC;
CTNNB1:
forward, ACCCCATCTCTCTTCGTCCT,
reverse, CCCTCCTAACAGCCTCACTT;
U6: forward, CTC GCT TCG GCA GCA CA,
reverse, AAC GCT TCA CGA ATT TGC GT;
GADPH:
forward, AAATTGAGCCCGCAGCCTCCC,
reverse, GCGCCCAATACGACCAAATCCGT.
Transwell assay
Transwell assays were performed according to the manufacturer’s instructions. Briefly, the cells (∼5 × 104) were harvested and resuspended in serum-free medium and then added to the Transwell upper chambers, in which the upper surface of the 8-μm pore size membrane (Corning, Steuben, New York, USA) was coated without or with Matrigel (BD Biosciences, Waltham, Massachusetts, USA). After incubation for 24 h, the cells that had migrated through the membrane were fixed, stained, and counted with an inverted microscope.
Immunohistochemistry
Immunohistochemical staining was performed as described previously[7]. Briefly, sections were blocked for endogenous peroxidase by incubation in 3% H2O2 for 20 minutes and then washed in PBS containing 0.05 M ethylenediamine tetraacetic acid (EDTA) followed by 4% paraformaldehyde. Then, the sections underwent heat‐induced antigen retrieval for 20 minutes in 0.01 M citrate buffer (pH 6.0) in the microwave. Tissues were blocked with Blocking Serum from the ABC Vectastain Kit (Vector Labs,
Burlingame, CA) for 20 minutes to block nonspecific binding. Then, 4 μm sections were incubated overnight at room temperature with anti-E-cadherin (Abcam) and anti-β-catenin antibodies (CST) in the presence of 10% rabbit serum. After the sections were washed, they were incubated for another 2 h with horseradish peroxidase (HRP) goat anti‐rabbit IgG secondary antibody. Slides were dehydrated and examined under a light microscope.
Western blotting
Western blotting was performed as described previously[7]. Briefly, 50 µg of protein lysate was loaded onto each well and resolved and then transferred to polyvinylidene fluoride (PVDF) membranes. The transferred membranes, blocked with 5% milk, were incubated overnight with primary antibodies against E-cadherin (Abcam) and β-catenin (CST) and washed with Tris‐buffered saline with Tween‐20 (TBST) (10 minutes, three times). The membranes were then probed with the appropriate secondary antibody (1:5000; Abcam). Immunoreactivity was determined and observed using enhanced chemiluminescence (Millipore, Billerica, MA, USA). β‐Actin was used as a control. ImageJ was used to calculate the relative expression of E-cadherin and β‐catenin at the protein level.
Immunofluorescence
Primary cells were washed with PBS and then fixed in 75% ethanol for 20 minutes at room temperature. After the cells were washed with PBS, they were blocked with 5% bovine serum albumin (BSA) for 1 h at 37°C. The cells were then incubated with a monoclonal anti‐human E-cadherin (Abcam) and β-catenin antibody (CST) overnight at 4°C. The cells were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, 10 µg/mL, 32670; Sigma‐Aldrich) to mark the nuclei. The cells were examined with a confocal laser scanning microscope.
Statistical analysis
All data are presented as the mean ± SEM. All experiments were performed at least three independent times. Using one‐way analysis of variance followed by the Duncan’s multiple‐comparison test using SPSS 19.0 (SPSS Inc., Chicago, IL), we calculated the statistical significance. P < 0.05, P < 0.01, or P < 0.001 was regarded as statistically significant.