Human tissue samples
Human glioma tissues and normal brain tissues (NBTs) were acquired from patients diagnosed with glioma undergoing neurosurgeries at Shengjing Hospital of China Medical University. All patients voluntarily signed informed consent before surgery and the consent was permitted by the Ethics Committee of Shengjing Hospital of China Medical University. Surgical resection specimens were placed in liquid nitrogen immediately after isolation before use. The pathological grades were determined by the pathologists according to WHO classification and were divided into low-grade glioma (WHO I-II: n = 15) and high-grade glioma (WHO III-IV: n = 15). Normal brain tissues acquired from patients undergoing traumatic surgeries without any clear brain diseases before (n = 15) were used as negative controls.
Cell culture
The human glioma cell lines (U87 and U251) and human embryonic kidney cell line (HEK293T) were purchased from Shanghai Institutes for Biological Sciences Cell Resource Center. Dulbecco’s modified Eagle medium (DMEM)/high glucose with 10% FBS was used to culture U87 and HEK293T cells while DMEM/F12 medium with 10% FBS was used to culture U251 cells. Normal human astrocyte (NHA) cells (Catalog #1800, 1 million cells per vial) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and were cultured in RPMI-1640 medium. All the cells were maintained in a humidified incubator at 37 °C with 5% CO2.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from human tissue samples and HA, U87 and U251 cells using Trizol reagent (Life Technologies Corporation, Carlsbad, CA). RNA quality and concentration were measured via 260/280 nm absorbance with Nanodrop Spectrophotometer (ND-100, Thermo, Waltham, MA). SNHG20, FOXK1 mRNA, GAPDH mRNA concentration were detected using One-Step SYBR PrimeScript RT-PCR Kit (TakaraBio, Inc., Japan) in 7500 Fast RT-PCR System (Applied Biosystems, USA). The relative quantification (2-△△Ct) was calculated after the expression levels were normalized using GAPDH as endogenous control.
Western blotting
Total proteins were gained after cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology) for 30 min and were then centrifuged at 17,000 g at 4 °C for 45 min. Protein concentrations were determined by BCA protein assay kit (Beyotime Institute of Biotechnology, China). Protein samples were subjected to SDS-PAGE and electrophoretically transferred to PVDF membranes. After incubated in Tween-Tris-buffered saline (TTBS) containing 5% nonfat milk at room temperature for 2 h, membranes were then incubated with primary antibodies as follows: ZRANB2 (1:500, Proteintech, Rosemont, IL), FOXK1 (1:200, Santa Cruz, USA), MMP1 (1:500, Proteintech, Rosemont, IL), MMP9 (1:500, Proteintech, Rosemont, IL), VE-Cadherin (1:500, Affinity Biosciences, USA), GAPDH (1:10000, Proteintech, Rosemont, IL). After washed three times with TTBS, membranes were incubated with horseradish peroxidase conjugated secondary antibodies at room temperature for 2 h. Immunoblots were visualized with ECL (enhanced chemiluminescence) Kit (Beyotime Institute of Biotechnology, China) and scanned using ChemImager 5500 V2.03 software (Alpha Innotech, San Leandro, CA). Integrated density values (IDVs) were calculated using GAPDH as an internal control.
Cell transfections
Short-hairpin RNA against ZRANB2 (ZRANB2(−)), short-hairpin RNA against FOXK1 (FOXK1(−)), short-hairpin RNA against STAU1 (STAU1(−)), short-hairpin RNA against UPF1 (UPF1(−)), ZRANB2 full length plasmid (ZRANB2(+)) and their respective non-targeting sequence (negative control, NC) were synthesized (GenePharma, Shanghai, China). Short-hairpin RNA against SNHG20 (SNHG20(−)) and its respective non-targeting sequence (negative control, NC) were synthesized (GeneChem, Shanghai, China). SNHG20 full length plasmid (SNHG20(+)) and FOXK1 full length plasmid (FOXK1(+)) and their respective non-targeting sequence (negative control, NC) were synthesized (GeneScript, Piscataway, Nj, USA). FOXK1 sequence with assumptive SNHG20 binding site was amplified by PCR and cloned into pmirGLO Dual-Luciferase Vector (GenePharama, China) as wild type (FOXK1–3’UTR-Wt). The corresponding FOXK1 sequence without assumptive SNHG20 binding site (GenePharama, China) was cloned into pmirGLO Dual-Luciferase Vector as mutant type (FOXK1–3’UTR-Mut). Cells were seeded in 24-well plates (Corning, NJ, USA) and transfected using Lipofectamine 3000 reagent (Life Technologies Corporation, Carlsbad, CA) according to its instructions. The stable transfected cells were selected using G418 (0.4 mg/ml), puromycin (1 μg/ml) or hygromycin (500 μg/ml). The silencing and over-expressions efficiency were measured by qRT-PCR. To investigate the effect of ZRANB2 on glioma, cells were divided into five groups: control, ZRANB2(−)-NC, ZRANB2(−), ZRANB2(+)-NC and ZRANB2(+) groups. To investigate the effect of SNHG20 on glioma, cells were divided into five groups: control, SNHG20(−)-NC, SNHG20(−), SNHG20(+)-NC and SNHG20(+) groups. To explore whether SNHG20 mediated the regulation of ZRANB2 affecting the behaviors of glioma, cells were divided into five groups: control, ZRANB2(−) + SNHG20(−), ZRANB2(−) + SNHG20(+), ZRANB2(+) + SNHG20(−), ZRANB2(+) + SNHG20(+) groups. To investigate the effect of FOXK1 on glioma, cells were divided into five groups: control, FOXK1(−)-NC, FOXK1(−), FOXK1(+)-NC and FOXK1(+) groups. To explore whether FOXK1 mediated the regulation of SNHG20 affecting the behaviors of glioma, cells were divided into five groups: control, SNHG20(−) + FOXK1(−), SNHG20(−) + FOXK1(+), SNHG20(+) + FOXK1(−) and SNHG20(+) + FOXK1(+) groups.
Cell proliferation assay
Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology, China) assay was used to evaluate the proliferation of cells. Cells were seeded in 96-well plates at the density of 2000 cells per well. After 72 h, 10 μl CCK-8 solution was added and cells were incubated at 37 °C for 2 h. The absorbance was measured at 450 nm on the SpectraMax M5 microplate reader (Molecular Devices, USA).
Cell migration and invasion assay
Cells were resuspended in 100 μl serum-free medium at the density of 2 × 105 cells per milliliter and seeded in the upper chamber for migration (or precoated with 500 ng/ml Matrigel solution (BD, Franklin Lakes, NJ) for invasion) with 8 μm pore size polycarbonate membrane (Corning, NY, USA) while 600 μl medium containing 10% FBS was added to the lower chamber. 24 h later, cells migrated or invaded to the lower side of membrane surface were fixed and stained 20% Giemsa. Three random fields under a microscope were chosen to count cell numbers.
In vitro VM tube formation assay
24-well plates were coated with 300 μl Matrigel solution per well and placed at 37 °C for 30 min. Cells were resuspended and seeded with serum-free medium at a density of 3 × 105 cells per well in the Matrigel-coated wells and incubated at 37 °C for more than 24 h. Photos of cells were taken under the inverted microscope (Olympus, Tokyo, Japan). Numbers of VM tube structures in three random fields were counted.
CD34-periodic acid-schiff (PAS) dual-staining
5 μm formalin-fixed and paraffin-embedded tissue specimens underwent dewaxed in xylene, hydrated in gradient ethanol and boiled in EDTA antigen-unmasking solution. After cooled gradually at room temperature, the tissue specimens were incubated with peroxide, blocked with goat serum and incubated with CD34 primary monoclonal antibody (1:50, Proteintech, Rosemont, IL) at 4 °C for 16 h. After incubation with secondary antibody at 37 °C for 10 min, the tissue specimens were stained with DAB kit (MaiXin Biotech, China). Periodic acid solution, schiff solution and hematoxylin were used for the next PAS staining. VM density was counted under microscope in five random fields.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (CHIP) kit (Beyotime Institute of Biotechnology, China) was used according to the manufacturer’s protocol. 1 × 107 cells were cross-linked with 1% formaldehyde for 10 min and then treated with glycine at room temperature for 5 min. Cells were lysed in buffer solution containing PMSF and resuspended. Chromatin was digested by micrococcal nuclease. Immunoprecipitates were incubated with 3 μg anti-FOXK1 antibody (Santa Cruz, USA) or normal rabbit IgG and incubated with Protein G agarose beads at 4 °C overnight with gentle shaking while 2% lysates were used as input reference. DNA crosslink was reversed with 5 M NaCl and proteinase K at 65 °C for 2 h. Then the DNA was purified and amplified by PCR using the following primers: one of putative binding site of FOXK1 in MMP1 promoter using the primers 5′- TGAGTAAGATATCAGTCTTGACGCA - 3′ and 5′- GTGTCTCCCACCTTTCCCAC - 3′, generating a 101 bp product; the other putative binding site of FOXK1 in MMP1 promoter using the primers 5′- CCTAGCACCAAGGAGCGAAG -3′ and 5′- CCGGATGATGAAAAGGCTGG - 3′, generating a 107 bp product; control group of putative binding site FOXK1 in MMP1 promoter using the primers 5′- TGCTTGTCATAAGGGGTAAAGGA -3′ and 5′- ACCAAATCCTAGCAATGCCT -3′, generating a 177 bp product; the putative binding site of FOXK1 in MMP9 promoter using the primers 5′- AGATCACGCCACTGCACTC -3′ and 5′- CGGGCAGGGTCTATATTCACC - 3′, generating a 147 bp product; control group of putative binding site FOXK1 in MMP9 promoter using the primers 5′- GACAGCCCCAAGTGCCAATA -3′ and 5′- CCCCCACTTGCCATCAATG -3′, generating a 101 bp product; the putative binding site of FOXK1 in VE-Cadherin promoter using the primers 5′- GGATGACACAACTGGCCAGA -3′ and 5′- GACTCCAGCTCTAAGGTGCC - 3′, generating a 119 bp product; control group of putative binding site FOXK1 in VE-Cadherin promoter using the primers 5′- AGGAGTCCCAAGGAGAGCTT -3′ and 5′- TGATGGGGTCAGAATGGCTG -3′, generating a 141 bp product.
Reporter vector constructs and luciferase reporter assay
HEK293T cells were seed in 96-well plates and co-transfected with FOXK1–3’UTR-Wt (or FOXK1–3’UTR-Mut) reporter plasmid and SNHG20(+)-NC or SNHG20(+), respectively. The relative luciferase activities were measured 48 h after co-transfection using Dual-Luciferase reporter assay kit (Promega).
RNA immunoprecipitation
EZ-Magna RNA-binding protein immunoprecipitation kit (Millipore, USA) was used according to the manufacture’s protocol. 3 × 107 cell lysate was incubated with RIP buffer with magnetic beads conjugated with 5 μg human anti-Ago2 or normal mouse IgG (as negative control). Samples were incubated with Proteinase K and immunoprecipitated RNA was then isolated. The concentration of RNA was measured by spectrophotometer (NanoDrop, USA). The RNA samples were purified and analyzed by qRT-PCR.
Nascent RNA capture
Click-iT® Nascent RNA Capture Kit (Thermo Fisher Scientific, USA) was used to detect nascent according to the manufacture’s protocol. Briefly, nascent RNAs were marked with 0.2 mM 5-ethymyl uridine (EU) and the EU-nascent RNA was captured on magnetic beads for subsequent qRT-PCR.
mRNA stability assay
Cells culture medium were added 5 μg/ml actinomycin D (ActD, NobleRyder, China) to inhibit the de novo RNA synthesis. Total RNA was extracted at 0, 3, 6, 9, 12, 15 h and its concentrations were measured by qRT-PCR. The half-life of RNA was determined by its level at certain point of time compared with time zero.
Xenograft mouse model in vivo
For the vivo study, 4-week-old athymic nude mice (BALB/c) were purchased from the Cancer Institute of the China Academy of Medical Science and randomly divided into five groups: control, ZRANB2(−)-NC + SNHG20(−)-NC, ZRANB2(−), SNHG20(−) and ZRANB2(−) + SNHG20(−) groups. All the experiments with mice were conducted strictly in accordance with the Animal Welfare Act approved by the Ethics Committee of China Medical University.
For subcutaneous xenografts, 3 × 105 glioma cells were injected into the right flank area of nude mice. Tumor volumes were measured every 5 days until ultimate 45 days according to the estimate formula: volume (mm3) = length × width2/2. VM of nude mice xenograft model was detected by CD34-PAS dual-staining later. For survival analysis, 3 × 105 glioma cells were stereotactically infected to the right striatum of nude mice. Kaplan-Meier survival curve was applied for the survival analysis.
Statistical analysis
All descriptive data are expressed as mean ± SD. SPSS 19.0 software was used for statistical analysis with Student’s t-test or one-way ANOVA. Differences were considered statistically significant if P < 0.05.