Patients and samples
Tissue samples were obtained from 94 patients with primary glioma undergoing surgical resection at the Department of Neurosurgery, The First Affiliated Hospital of Sun Yat-Sen University, Guangdong, China from January 2010 to July 2014. No patients received chemotherapy or radiotherapy prior to surgery. Final diagnosis was confirmed by two independent pathologists and graded according to the 2016 World Health Organization grading system for central nervous system tumors. Four normal brain tissues were collected from patients with hernia during surgical decompression. All tissue samples were fixed in 4% neutral buffered formaldehyde at 4℃ followed by paraffin embedding. The inclusion of patients in this study was unbiased and was only dependent on the availability of tumor materials and clinical follow-up data. The follow-ups were terminated in July 2017. This study was approved by The Ethics Committee of Sun Yat-Sen University and was in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient.
Immunohistochemical (IHC) staining
Paraffin-embedded tissue sections (4-µm thick) were deparaffinized and rehydrated followed by IHC staining for glial fibrillary acidic protein (GFAP), Cav-1, or CD31. For antigen retrieval, the sections were heated in 1 mM EDTA (pH 8.0) for 15min. The sections were then blocked with goat serum followed by incubation with anti-GFAP (Abcam, Cambridge, MA, USA), anti-Cav-1 (Abcam, Cambridge, MA, USA), or anti-CD31 (Abcam, Cambridge, MA, USA) at 4°C overnight and with horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam, Cambridge, MA, USA) at room temperature for 30 min. Normal IgG was used as a negative control. Finally, the sections were stained with diaminobenzidine and the nuclei were counterstained with hematoxylin. An Olympus IX81 fluorescence microscopy (Olympus, Japan) was used for visualization and brown staining was considered positive. The results were evaluated by two independent pathologists in a blinded manner. Cav-1- or CD31-positive cells with brown staining were counted in five randomly selected fields in each section at 400× magnification. The mean number of Cav-1-positive cells was calculated (13 Cav-1-positive cells per field). Therefore, a mean number < 13 was considered low expression whereas a mean number ≥ 13 was considered high expression. Representative images (magnification ×400) were acquired using a XDS-100 Caikang microscopy (Caikang, Shanghai, China).
CD31/PAS double staining
Following IHC staining for CD31, the sections were exposed to 1% sodium periodate for 10 min, rinsed with distilled water for 5 min, and then incubated with PAS in dark at 37°C for 15 min. The sections were then counterstained with hematoxylin and the results were visualized at 400× magnification using an Olympus IX81 microscope. CD31 staining (brown) represents blood vessels in tissues, whereas CD31-negative/PAS-positive (light Purple) staining represents the wall of the VM channels. The number of VM channels was counted in randomly selected 5 fields in each section. The average number was calculated (1.4/field). VM number < 1.4 per field was defined as low, while ≥ 1.4 was defined as high.
Cell culture and treatments
Human malignant glioma cell lines U251 and human umbilical vein endothelial cells (HUVECs) were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science (CBTCCCAS; Shanghai, China). These cell lines was authenticated by DNA fingerprinting, isozyme detection and cross species checks. All cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Hyclone，Cat.No.SH30023.01B, Logan City, Utah, United States) supplemented with 10% fetal bovine serum (FBS; Hyclone，Cat.No.SH30087.01, Logan City, Utah, United States) at 37°C in a humidified incubator with 5% CO2.
Generation of glioma cell lines with Cav-1 silencing or overexpression
Small interfering RNA (siRNA) targeting Cav-1 (siCav-1) was purchased from Sigma (Shanghai, China). The sequences of siCav-1 were 5’-CCCUAAACACC UCAACGAUdTdT-3’ (sense) and 5’-AUCGUUGAGGUGUU UAGGGdTdT-3’ (antisense). The sequences of negative control siRNA (siNC) were 5’-UUCUCC GAACGUGUCACGUTT-3’ (sense) and 5’- ACGUGACACGUUCGGA GAATT-3’ (antisense).
The coding sequence of human Cav-1 was amplified using the primers 5'-ccgctcgagATGTCTGGGGGCAAATACGTAG-3' (forward) and 5'-cggggtaccTTA TATTTCTTTCTGCAAGTTGATGC-3' (reverse), and was inserted into the XhoI and KpnI sites of linearized pEGFP-C3 vector (Clontech Laboratories，CA，USA) according to the manufacturer's instructions. The recombinant expressing vector pEGFP-C3-Cav-1 was sequenced by BGI Corporation (Shenzhen, China) to confirm the cloned sequences.
U251 cells were seeded in a six-well plate at a density of 5 ×105 cells/well and grown overnight. pEGFP-C3-cav-1 or siCav-1 was transfected into U251 cells using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Empty vector pEGFP-C3 and the vector expressing siNC were used as negative controls. After 48–72 h of transfection, the mRNA level of Cav-1 was determined using quantitative real-time PCR (qPCR).
Tubule formation assay
Tubule formation was evaluated using a Matrigel (BD Biosciences, CA, US)-based three-dimensional (3D) culture model. Briefly, 50 μL of Matrigel was added into each well of 96-well plates and allowed to polymerize at 37°C for 30 min. HUVECs or U251 cells were trypsinized, resuspended in serum-free medium, and seeded onto the Matrigel layer at a density of 2 × 104 cells per well. After 6 h of incubation at 37°C, the images of each well were captured using an Olympus BX61 phase-contrast fluorescence microscope (magnification ×100). Four randomly selected areas of vascular network meshes in each well were measured using Image J software (NIH, Bethesda, MD, USA). All experiments were performed in triplicate.
RNA extraction and qPCR
Total RNA was isolated from tissues or cells using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The residual DNA was removed using DNase I (Roche, Indianapolis, IN, USA). 2 μg of RNA was reversely transcribed to synthesize cDNA using a M-MLV Reverse Transcriptase Kit (Thermo Fisher, Rockford, IL, USA). The cDNA was amplified using SYBR Green qPCR Master Mix (Thermo Fisher) in an ABI 7300 system (Applied Biosystem, Foster City, CA, USA) following the manufacturer’s instruction. The relative mRNA level of Cav-1, HIF-1α, or Akt was calculated by normalization to that of 18s. The PCR primers were as follows: Cav-1, 5’-CACCTAAGCTGCACAGTTCC-3’ (forward) and 5’-GGCT GCCTCCTAATTCTTCC-3’ (reverse); 18s, 5’-CCTGGATACCG CAGCTAGGA-3’ (forward) and 5’-GCGGCGCAATACGAATGCCCC-3’ (reverse); AKT1, 5’-ATCGCT TCTTTGCCGGTATC-3’ (forward) and 5’-CTTGGTCAGGTGGTGTGATG-3’ (reverse); HIF-1α 5’-GTGGATTACCACAGCTGA-3’ (forward) and 5’-GCTCA GTTAACTTGATCCA -3’(reverse).
Western blot analysis
Cell lysates were obtained from U251 cells or grounded tissue samples using RIPA buffer containing 1% phenylmethylsulfonyl fluoride. The protein concentration was determined using a BCA protein assay kit (Beyotime). Equal amounts of total protein were separated in 10% SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk for 1 hour followed by an incubation with primary antibody against Cav-1, Akt, HIF-1α or GAPDH (Abcam, Cambridge, MA, USA) at 4°C over night. The membranes were then incubated with HRP-linked secondary antibodies (1:5,000; Beyotime) for 1 h. The protein bands were visualized using enhanced chemiluminescence assay (Thermo) and exposed to X-ray films. The results were scanned and quantified using Image J software.
Cell proliferation assay
Cell proliferation assay was carried out using cell counting kit-8 (CCK-8; Dojindo Laboratories, Japan) following the manufacturer’s protocol. Briefly, U251 cells were seeded into 96-well plates at a density of 2000 cell/well and transfected with siRNA. 10 μL of CCK-8 solution was added into each well at 0, 24, 48 or 72 h after transfection followed by an incubation at 37 °C for additional 1 h. The absorbance was measured at 450 nm using a microplate reader (Bio-Rad).
Statistical analysis was performed using SPSS 22.0 (SPSS, IL, USA) or GraphPad Prism 6 (GraphPad, CA, USA). The association between CAV-1 and VM was examined by Spearman analysis, and the differences of clinicopathological variables were analyzed with χ2-test. Statistical comparisons among groups were performed using Student’s t test and ANOVA. Data were expressed as the mean ± standard error of the mean. All experiments were repeated independently at least three times. P < 0.05 was considered statistically significant.