Human brain tumor (n = 68; WHO grade II–IV) and non-neoplastic tissue (n = 4) samples were obtained from surgeries performed at the Department of Neurosurgery at Qilu Hospital (Shandong, China). Written informed consent was obtained from all patients, and approval for experiments was obtained from Ethics Committee of the Qilu Hospital. All surgeries and post-operative animal care were approved by the Institutional Animal Care and Use Committee (IACUC) of Shandong University (Shandong, China). Our research complies with the commonly-accepted ‘3Rs’: replacement of animals by alternatives wherever possible, reduction in the number of animals used, and refinement of experimental conditions and procedures to minimize harm to animals.
Cell culture and hypoxic treatment
Human glioma cell lines, U87MG, U251 and T98 were obtained from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The normal human astrocytes (NHA) cell line was a kind gift from the Department of Biomedicine at the University of Bergen (Bergen, Norway). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and maintained at 37 °C in a humidified chamber containing 5% CO2. For hypoxic treatment, cells were placed in a modulator incubator (HERAcell 150i, Thermo Fisher Scientific) in 94% N2, 5% CO2, and 1% O2. For stable CCDC109B-knockdown, U87MG and U251 cells were infected with lentivirus expressing short hairpin RNA (shRNA) (sh-CCDC109B-1). After 48 h, U87MG or U251 cells were exposed to 0.5 or 2 µg/mL puromycin (A1113802, Thermo Fisher Scientific), respectively, in complete DMEM for an additional 2 weeks. Cells were subsequently treated with PX478 (S7612, Selleck Chemicals; Shanghai, China) and HIF1α siRNA to inhibit HIF1α expression and harvested after 48 h. Sequences of synthesized shRNAs (Genepharma; Shanghai, China) were the following: sh-Negative Control (sh-NC) 5′-TTCTCCGAACGTGTCACGTtt-3′; sh-CCDC109B-1 5′-CAGTCACACCATTATAGTAtt-3′; sh-CCDC109B-2 5′-CTCGACAGGATTATACTTAtt-3′; sh-CCDC109B-3 5′-GCAAGTAGAAGAACTCAATtt-3′. Sequences of synthesized siRNAs (Genepharma) were the following: si-NC 5′-TTCTCCGAAGGTGTCACGG-3′; si-HIF1α-1 5′TACGTTGTGAGTGGTATTATT-3′; si-HIF1α-2 5′-CTGATGACCAGCAACTTGA-3′.
Samples were fixed in 4% formalin, paraffin-embedded, and sectioned (4 µm). After de-waxing and rehydration, the sections were incubated with 0.01 M citrate buffer for 20 min at 95 °C for antigen retrieval. Endogenous peroxidase activity and non-specific antigens were blocked with 3% hydrogen peroxide (ZSGB-Bio; Beijing, China) and 10% normal goat serum (ZSGB-Bio) respectively, followed by incubation with primary antibody at 4 °C overnight. Sections were rinsed with phosphate buffered saline (PBS), treated with goat anti-rabbit secondary antibody (ZSGB-Bio), visualized using 3, 3′-diaminobenzidine (DAB, ZSGB-Bio) as substrate, and counterstained with hematoxylin (Beyotime; Haimen, China). Normal mouse serum was used as the negative control. Staining of cancer cells was scored as follows: 0, no staining; 1, weak staining in <50% cells; 2, weak staining in ≥50% cells; 3, strong staining in <50% cells; and 4, strong staining in ≥50% cells. The following primary antibodies (Abcam, Cambridge, UK) were used at the dilutions indicated: CCDC109B (1:200), HIF1α (1:200), Ki-67 (1:500), MMP2 (1:100) and MMP9 (1:200).
Western blot analysis
Cells and tissues were incubated 30 min in RIPA buffer containing protein inhibitor cocktail for lysis (Thermo Fisher Scientific). After centrifugation and denaturation, protein (20 μg) was separated by 10% polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore; Shanghai, China). Membranes were blocked with Tris Buffered Saline with Tween 20 (TBST, 10 mM Tris, 150 mM NaCl, 0.1% Tween 20) containing 5% bovine serum albumin (BSA, Thermo Fisher Scientific),and incubated overnight at 4 °C with the following primary antibodies against CCDC109B (1:500), HIF1α (1:1000), MMP2 (1:1000), MMP9 (1:1000) and β-Tubulin (1:1000; Cell Signaling Technology; Danvers, MA, USA). Membranes were incubated the next day with secondary antibody (1:5000; Santa Cruz; Dallas, TX, USA) conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. Proteins were quantified using a system for detecting chemiluminescence (Bio-Rad; Irvine, CA, USA), according to the manufacturer’s protocol. Representative images and data were obtained from at least three independent biological replicate experiments.
Cell migration and invasion assay
Cell migration and invasion assays were performed in uncoated and matrigel-coated (BD Biosciences; San Jose, CA, USA) Transwell chambers (8 μm pores; Corning Costar; Corning, NY, USA). Cells (2 × 104) in medium (200 µL) with 1% FBS were seeded in the top chamber. The lower chamber was filled with medium (600 µL) containing 30% FBS. Chambers were incubated for 24 h under normoxic or hypoxic conditions. Cells that migrated to or invaded into the lower surface were fixed with 4% paraformaldehyde (Solarbio; Beijing, China), stained with crystal violet (Solarbio) for 15 min and counted under bright field microscopy. Images were acquired from 5 random fields in each well, and cell numbers were determined using Kodak MI software. Each experiment was repeated three times in triplicate.
To assess the distribution and expression levels of CCDC109B, NHA and glioma cells were seeded onto glass slides. The cells were then washed twice with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were rinsed with PBS, permeabilized with 0.5% Triton X-100 (Solarbio) for 15 min, and blocked with 10% normal goat serum for 60 min at room temperature. Cells were stained with primary antibody against CCDC109B (1:100) at 4 °C overnight, followed by incubation with Alexa Fluor 594 goat anti-rabbit IgG (Abcam, UK; 1:800) for 1 h at room temperature. Cell nuclei were stained with DAPI (Sigma-Aldrich, Germany) at 37 °C for 10 min, and images were obtained with confocal microscopy (LSM780, Zeiss).
Cell proliferation was measured using the EdU Apollo 567 Cell Tracking Kit (Ribo-bio; Guangzhou, China). Cells (2 × 104) under different treatments were seeded onto 24-well plates, exposed to 200 μM of 5-ethynyl-20-deoxyuridine for 2 h at 37 °C, fixed with 4% paraformaldehyde for 20 min, and treated with 0.5% Triton X-100 for 10 min. Cells were rinsed with PBS three times, and incubated with 100 μL of Apollo reagent for 30 min. Nuclie were stained with Hochest33342. The percentages of EdU-positive cells were determined from 500 cells and three independent experiments were performed.
Plate colony forming assay
NC and sh-CCDC109B-1 glioma cells were seeded onto six-well plates (120 cells per well) and cultured for 2 weeks in medium that was changed twice each week. Colonies of more than 50 cells were counted after fixation and staining with 100% methanol and 5% crystal violet. Data reported represent the average of three independent experiments.
Quantitative real-time PCR
Total RNA was isolated from cells using Trizol reagent (Takara; Tokyo, Japan) according to the manufacturer’s protocol. Total RNA was reverse-transcribed, and the resulting cDNA was used as template in real-time quantitative PCR performed with the standard SYBR premix Ex Taq (Takara) on the Real Time PCR Detection System (480II, Roche; Pleasanton, CA, USA). GAPDH served as an internal control, and independent experiments were conducted in triplicate. The following primers were used: GAPDH, forward, 5′-AATGAAGGGGTCATTGATGG-3′, reverse, 5′-AAGGTGAAGGTCGGAGTCAA-3′; HIF1α, forward, 5′-TGGCAGCAACGACACAGAAA-3′, reverse, 5′-TGCAGGGTCAGCACTACTTC-3′; CCDC109B, forward, 5′-ACACTGCTGAGATGGAACACAT-3′, reverse, 5′- TTGGCTTCCGAATGAGCTTCTA-3′.
For generation of the subcutaneous GBM model, female 4-week-old nude mice (SLAC laboratory animal Center; Shanghai, China) were maintained in a barrier facility on high-efficiency particulate air (HEPA)-filtered racks. Digoxin and saline were purchased from Qilu Hospital, Shandong University. Nude mice (n = 16) were divided into two groups (U87MG + saline, U87MG + digoxin, 8 mice per group). Cells were harvested by trypsinization, resuspended at 107 cells/mL in a 1:1 solution of PBS/Matrigel (BD Biosciences, USA), and injected subcutaneously into the right shoulder of the mouse. The tumor tissues were isolated 37 days after injection, and then used for protein extraction.
For orthotopic xenografts, 4-week-old female nude mice (n = 16) were divided into two groups (sh-CCDC109B-1 and NC group), and U87MG or U87MG modified cells (1 × 106) were implanted into the brain using a stereotactic apparatus (KDS310, KD Scientific; Holliston, MA, USA). Animals which displayed symptoms such as severe hunchback posture, apathy, decreased motion or activity, dragging legs, or drastic loss of body weight were euthanized by cervical dislocation. Excised tumor tissues were formalin-fixed, paraffin-embedded, and sectioned for Hematoxylin–Eosin (HE) staining and IHC.
All data are presented as a mean ± the standard error of the mean (S.E.M). The Student’s t test was used when only two groups were being compared. Analysis of variance (ANOVA) was used in cases where there were more than two groups being compared. Survival curves were estimated by the Kaplan–Meier method and compared using the log-rank test. For multivariate analysis, independent prognostic factors were determined using the Cox’ proportional hazards model. Variables that might be dependent on other variables were excluded from the model. A two-tailed χ2 test was used to determine the association between CCDC109B and HIF1α. GraphPad Prism version 7.00 software program (GraphPad; La Jolla, CA, USA) was used to analyze in vitro and in vivo experiments. Differences were considered to be statistically significant when P < 0.05.