Patient samples
72 GBM tissues, 41 low grade glioma (LGG) tissues and 15 normal brain tissues (NBT) applied in this study were obtained from the Department of Neurosurgery, the Second Affiliated Hospital of AnHui Medical University from January 2013 to October 2018. All patients signed the written informed consent, and this study was reviewed and approved by the Clinical Research Ethics Committee at the Second Affiliated Hospital of AnHui Medical University.
Cell culture
Human GBM cell lines (U251, LN18, U87 and A172) were obtained from the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone, Logan, UT, USA) with high glucose supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and streptomycin (100μg/ml), penicillin (100 U/ml). Normal human astrocyte (HA) was purchased from Sun Yat‑Sen University and cultured in astrocyte medium with low glucose supplemented with 10% fetal bovine serum. All cell lines were cultured at 37 °C in a humidified incubator with 5 % CO2.
Subcellular fractionation
Cytoplasmic and nuclear fractions of the LN18 cells were prepared and collected according to the manufacturer’s instructions of PARIS™ Kit (Ambion, USA). GAPDH was used as the cytoplasmic endogenous control. U6 small nuclear RNA was used as the nuclear endogenous control.
Cell transfection
SiRNA targeting HNF1A-AS1 (si-HNF1A-AS1), EGR1 (si-EGR1) and scrambled negative control (si-NC) were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The sequence of si-HNF1A-AS1-1 was as follows: CCCTCCATCTAACATTCAA, si-EGR1, CAACGAGAAGGTGCTGGTG, and these fragments were transfected into U251 and LN18 cells respectively by using Lipofectamine2000 (Invitrogen, USA) according to the manufacturer’s protocols. full‐length of HNF1A‐AS1 and EGR1 was amplified by PCR and sub‐cloned into pcDNA3.1-Vector (pcDNA3.1-HNF1A-AS1 and Vector), pcDNA3.1-Vector (pcDNA3.1-EGR1 and Vector) (Sangon Biotech, Shanghai). MiR-22-3p negative control (NC), miR-22-3p mimics and miR-22-3p inhibitors were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China), and transfected into cell lines as the above described.
Total RNA isolation and quantitative Real-time PCR (RT–QPCR)
Total RNA was isolated from cultured cells or tissues with ready to use TRIzol Reagent (Invitrogen, USA), according to the manufacturer's instructions. Using a Nanodrop Spectrophotometer (IMPLEN GmbH, Munich, Germany), RNA concentration and quality were determined by the 260/280 nm absorbance. Then RNA was reversely transcribed into cDNA by using PrimeScript™ RT Master Mix (Perfect Real Time) (TaKaRa Biotechnology, Dalian, China). Maxima SYBR‑Green/ROX qPCR Master Mix (Thermo Fisher Scientific, Inc.) was used for quantitative PCR. The primers for genes were determined as follows: HNF1A-AS1 forward 5′- CAAGAAATGGTGGCTATGA-3′, reverse 5′- TGGACTGAAGGACAAGGGT-3′;
EGR1, forward: 5’-CAGCACCTTCAACCCTCAG-3′, reverse: 5’-CACAAGGTGTT
GCCACTGTT-3′; ENO1 forward 5′- GCCTCCTGCTCAAAGTCAAC-3′, reverse 5′- AACGATGAGACACCATGACG-3′; GAPDH forward 5′-AGCAAGAGCACAAGAGGAAG-3′, reverse 5′-GGTTGAGCACAGGGTACTTT-3′. All‑in‑One™ miRNA First‑Strand cDNA Synthesis kit (Genecopoeia, Guangzhou, China) was used for miRNA reverse transcription and RT–QPCR was conducted using the All‑in‑One™ miRNA qPCR kit (Genecopoeia) for miR‑204‑3p and U6 (miRQ0022693‑1‑1/MQP‑0202, respectively; RiboBio Co., Ltd.), respectively, using ABI 7300 (Applied Biosystems, Darmstadt, Germany). GAPDH and U6 were used as loading control for HNF1A-AS1, ENO1 and miR-22. PCR Cycling conditions for mRNA was 2 min at 50 °C, 10 min at 95 °C and followed by 45 cycles of 95 °C for 15 s, 60 °C for 60s; for miRNA was 10 min at 95 °C and followed by 45 cycles of 95 °C for 10s, 60 °C for 20s and 72 °C for 12 s. All RT–QPCR reactions were performed in triplicate. The data were determined using the 2−△△Ct method.
Cell proliferation assay
GBM cells were placed into 96-well plates at the density of 2000 cells/well. Approximately 20 μl of CCK8 regent was added to each well after transfection. Finally, the absorbance at 450 nm was measured using a ST-360 micro-plate reader (KHB, Shanghai, China) after incubated at 37 °C for 2 h.
Colony formation assay
For the clone formation assay, 48 hours after transfection, GBM cells (200 viable cells per well) were seeded in a 6-well plate and cultured with complete medium for 12 days. cells were fixed with 4 % polyoxymethylene and stained with 1.5 % methylene blue for 30 min at room temperature.
Wound healing, migration and invasion assays
For the wound healing assay, 48 hours after transfection, GBM cells (200 viable cells per well) were seeded in a 6-well plate and cultured with complete medium. a 10-μl pipette tip was used to create wound gaps, gently washed, and cultured with serum-free medium for 24 h. The wound gaps were observed at 0 and 24 h after wounding and photographed with a light microscope (IX71; Olympus, Tokyo, Japan) at × 200 magnification. Lines were drawn along the leading edges of the cells, and the gap distances of migrating cells from five different areas for each wound were measured and analyzed.
GBM Cells migration and invasion ability was tested by using 24-well chambers with 8 μm pore size (Corning, USA). A total of 2x104 cells were resuspended in 150 μl serum‑free medium and seeded into the upper chamber with or without pre‑coated with 500 ng/ml Matrigel solution (BD Biosciences, San Jose, CA, USA), while 500 μl of 10 % FBS medium was placed in the lower chamber of Transwell plates, following incubation at 37˚C for 48 h for the migration and invasion assays. Non‑migrated and non‑invaded cells from the upper chamber were scraped off using a cotton swab. The migrated and invaded cells on the lower chamber membrane were fixed with 4% polyoxymethylene and stained with crystal violet (Sigma) at room temperature for 5 min, and dried. Five predetermined fields were counted under a microscope (Olympus IX71, (Olympus; x200 magnification). All assays were performed in triplicate.
Luciferase reporter assays
The fragments of HNF1A-AS1 and 3′ UTR of ENO1, both containing the predicted miR-22 binding site, then the predicted wild-type (WT) binding sites of miR-22 and mutant binding sites (Mut) were cloned into a pmiRGLO Dual-luciferase miRNA Target Expression Vector (Promega, Madison, WI, US), termed as pmiRGLO-HNF1A-AS1-wild-type (HNF1A-AS1-WT), pmiRGLO-HNF1A-AS1-mutated-type (HNF1A-AS1-Mut), pmiRGLO-ENO1-wild-type (ENO1-WT) and pmiRGLO-ENO1-mutated-type (ENO1-Mut). Then HNF1A-AS1-WT or HNF1A-AS1-Mut was co-transfected with the miR-22 mimics or miR-22 NC into GBM cells by using Lipofectamie2000 (Invitrogen, USA). After 48 h transfection for luciferase assay using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. ENO1-WT and ENO1-Mut were handled similarly as described above.
To confirm the bind relation between EGR1 and HNF1A-AS1 promoter, pGL3-HNF1A-AS1 promoter was co-transfected into cells along with si-EGR1 or si-NC using Lipofectamine 2000. The luciferase activity was measured by a Dual-Luciferase reporter assay system (Promega, USA). All assays were independently performed in triplicate.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assays were performed using the EZ-Magna ChIP™ Chromatin Immunoprecipitation Kit (Millipore, Darmstadt, Germany). U251 and LN18 cells were fixed with 1% formaldehyde for 10 min at room temperature and lysed in ChIP lysis buffer, and then the DNA was sonicated for shearing DNA into 500-bp fragments. DNA samples were precipitated with anti-IgG or anti-EGR1 antibody and Protein A/G magnetic beads for overnight. Subsequently, the co-precipitated chromatin DNA was collected, and was tested by RT-QPCR.
RNA immunoprecipitation
RNA immunoprecipitation (RIP) experiments were performed by the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA), and following the manufacturer's protocol. GBM cells were lysed by RIP lysis buffer, and magnetic beads conjugated with human anti-Argonaute2 (Ago2) antibody (Millipore) or normal mouse IgG (Millipore), which was indicated as negative control. Samples were incubated with Proteinase K buffer and then immunoprecipitated RNA was isolated. Furthermore, purified RNAs were extracted and assayed by RT-QPCR.
Western blotting
Western blotting was performed as described previously (Ma et al., 2016). Briefly, cells were collected and lysed using RIPA protein extraction reagent (Beyotime, Shanghai) with a protease inhibitor cocktail (Beyotime), on ice, subjected to SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes, which were then blocked in buffer (5% nonfat milk in TBST) about 1.5h before being incubated with primary antibodies (anti-rabbit-ENO1, 1:1000, Abcam, EUGENE, USA), (anti-rabbit-EGR1, 1:1000, CST, USA) and anti-rabbit-β-actin (1:1000, Abcam, EUGENE, USA) at 4 °C overnight. Horseradish peroxidase-conjugated goat anti-rabbit (1:5000, Beyotime) was applied as a secondary antibody and incubated at 4°C for 1h, and then immune complexes were visualized by SuperSignal ® West Femto Trial Kit (Thermo Fisher, USA) and blot bands were scanned using Find-do × 6 Tanon (Tanon, Shanghai, China).
Tumor xenograft model
Female nude mice at 4–6 weeks of age were used in this study, and experiments with nude mice were conducted strictly in accordance with a protocol approved by the Administrative Panel on Laboratory Animal Care of the Second Affiliated Hospital of AnHui Medical University. The animals were free to autoclaved food and water during the study. U251 cells stably transfected with si-NC or si-HNF1A-AS1 were collected, and injected into the subcutaneous tissues of the axillary skin. The tumor volumes were measured every five days after inoculation. 40 days after injection, the mice were sacrificed, and the tumor nodules were harvested for further study.
Statistical analysis
Unless stated otherwise, all experiments were performed in triplicate and all data were presented as the mean ± standard deviation (SD). GraphPad Prism V6.01 (GraphPad Software, Inc., La Jolla, CA, USA) software was used for statistical analysis and generate figures. Differences were analyzed by SPSS statistical software (version 19.0, Armonk, NY, USA) with the Student’s t-test or one-way ANOVA. Pearson’s correlation was performed to analyze the relationship between the expression of HNF1A-AS1, miR-22 and ENO1 in tissues. Survival analysis was performed using the Kaplan-Meier method and log-rank tests in GraphPad Prism 6.01. Differences were considered significant if P < 0.05.