Patients and clinical samples
In the study, the cohort of tissue specimens contained 106 glioma tissues and 23 normal brain tissues obtained from the Department of Neurosurgery of the First Affiliated Hospital of USTC between February 2014 and September 2019. All tumor samples were clinicopathologically confirmed as glioma (12 Grade I, 15 Grade II, 30 Grade III and 49 Grade IV). All of glioma tissues were astrocytoma and IDH wildtype. Normal brain tissues were collected from patients undergoing brain tissue resection due to craniocerebral injury. All samples were immediately stored in liquid nitrogen with RNAhold (TransGen). Written informed consent was obtained from all patients, and the study was approved by the Ethics Committee of the First Affiliated Hospital of USTC (2016-ky072). The basic characteristics of the included patients are shown in Table 1.
RNA extraction and transcriptome data analysis
Samples (three glioblastoma tissues and three corresponding normal brain tissues) were used to extract total RNA by using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The RNA quality was measured with a NanoDrop ND-3300 and verified by gel electrophoresis. Ribo-minus transcriptome libraries were constructed with the TruSeq Ribo Profile Library Prep Kit (Illumina) according to the manufacturer’s protocols. The libraries were then subjected to 151-nt paired-end sequencing, generating a depth of ~ 100 million read pairs, with an Illumina Nextseq 500 system (Novogene). Adapters were first trimmed with cut adapt to obtain clean reads and the remaining reads were aligned to the human genome (hg19) with bowtie2, with one mismatch allowed. Continuous or non-continuous mapped reads were subjected to the following mRNA and lncRNA analyses. Linear expression levels were evaluated with TopHat2 and Cufflinks followed by the annotation references of Refseq. Differentially expressed lncRNAs were identified using strict filtering criteria (| log2(fold change) | ≥ 1.5 and P value < 0.01), and (| log2(fold change) | ≥ 1 and P value < 0.01) for mRNAs.
Gene expression profile sets GSE50161 and GSE33331 [43, 44] were downloaded from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) [45]. The two datasets were based on GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array. The two datasets were merged to research the expression trend of lncRNAs. GEPIA (Gene Expression Profiling Interactive Analysis) (http://gepia.cancer-pku.cn) [46], a web-based tool that delivers fast and customizable functionalities based on TCGA and GTEx data, was employed to further verify the expression profile of lncRNAs.
Bioinformation analysis
The Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) is widely applicable to systematic analysis of gene functions [47]. Database for annotation, visualization, and integrated discovery (DAVID) is an analytical tool that is used for integrative analysis of large gene lists [48]. In this study, we used DAVID (version 6.8) to perform KEGG pathway enrichment analyses for differentially expressed genes with the following cutoff thresholds: enrichment gene number > 2 and P value < 0.05.
Cell lines and culture conditions
Human glioma cell lines (U251, U87, SHG44, T98G, GOS-3, TJ905, U373) and normal cells (HEB) were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and maintained in our lab. All cell lines underwent a mycoplasma contamination test and determined to be mycoplasma-free. All cells were cultivated in high-glucose Dulbecco’s Modified Eagle Medium (DMEM, HyClone) containing 10% FBS (Clark) and stored in an incubator with 5% CO2 at a constant temperature of 37 °C.
RNA extraction and PCR
Total RNA from glioma tissues and cell lines was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocols, and 1μg of RNA quantified by a NanoDrop ND-3300 (Thermo Fisher Scientific) was reverse transcribed using GoScript Reverse Transcription System (Promega) with corresponding primers. Real-time PCR analyses were performed with TransStart Top Green qPCR SuperMix (+Dye II) (TransGen) on an ABI Q5 Sequence Detection system (Applied Biosystems); GAPDH was used as an internal control. Bulge-Loop miRNA-specific Primer (RiboBio) was applied to measure miR-7-5p expression according to the manufacturer’s synopsis, and U6 was used as an endogenous control. Relative mRNA and miRNA expression levels were analyzed using the 2-ΔΔCt method. All primers were synthesized by Sangon Biotech; detailed information is shown in Table S1.
Nuclear-cytoplasmic fractionation
Nuclear/cytoplasmic fractionation was performed with a Nuclei Isolation Kit (KeyGEN BioTECH) according to the manufacturer’s protocols. Nuclear and cytoplasmic RNA was analyzed by real-time quantitative PCR; U6 was used as the nuclear fraction control, while GAPDH served as the cytoplasmic fraction control.
Plasmids, siRNAs, and transfection
For LPP-AS2 and EGFR overexpression, full-length LPP-AS2 and EGFR cDNA was amplified and subcloned into pEGFP-C1; the empty vector was used as a negative control. All plasmids were isolated using Endo-free Plasmid DNA Mini Kit I (OMEGA). SiRNAs, miRNA mimics and inhibitors were all obtained from RiboBio. All siRNAs were BLAST searched to ensure that no more than 17-nt matches occurred in the corresponding genomes [49]. SiRNA and plasmid transfection was conducted with Lipofectamine 3000 reagent (Invitrogen) or lipo8000 reagent (Beyotime) in accordance with the manufacturer's protocol.
Lentiviral vector construction and stable transfection
Lentiviral constructs of sh-LPP-AS2 was conducted by Hanbio Biotechnology and constructed into SHG44 cell lines. Cells were transfected with lentivirus or negative control virus (NC) in order to select the stably transfected cells. The cells were then treated with puromycin (2 μg/mL) (Solarbio) for two weeks. GFP-positive cells were selected as sh-LPP-AS2 and sh-NC stably transfected cells and validated by real-time quantitative PCR.
Tumor xenograft model
Female BALB/c nude mice (aged 4–5 weeks, 18–20 g) were purchased from Vital River Laboratory Technology, and reared in laminar airflow cabinets under specific pathogen-free conditions. Subsequently, 1×107 cells stably transfected with sh-LPP-AS2 or sh-control were suspended in 0.1 mL PBS and 0.1 mL Matrigel substrate and injected subcutaneously into the armpit regions of the mice. Tumor volumes were measured every 3 days and calculated using the following formula: volume (cm3) = (length × width2)/ 2. Bioluminescent imaging was performed using IVIS Lumina LT Series Ⅲ Imaging System (IVIS Lumina) with administration of D‐luciferin (150 mg/kg i.v.). The mice were sacrificed after 18 days post-injection, and the tumors were gathered for subsequent analysis. The animal studies were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of USTC.
RNA pull-down with biotinylated antisense oligonucleotides
RNA pull-down with 5′-biotinylated AS oligos was used a previously described method [50]. Cells were cross-linked in a UV cross-linker (UVP) at a strengthen of 200-mJ. The cells were granulated and resuspended in RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% SDS, 1 mM DTT, 1× protease-inhibitor cocktail (Roche), and 0.1 U/µL RNase inhibitor) for 10 min on ice and then harvested and sonicated for 10 min. Cell debris in the lysate was removed by centrifugation at 13,000 g for 20 min. Subsequently, biotinylated AS oligonucleotides (100 pmol) or Scramble oligos (as control) were added to the supernatant at 4 °C for 2 h. M-280 Streptavidin Dynabeads (Thermo) were washed three times in RIPA buffer and then blocked with 500 ng/µL yeast total RNA and 1 mg/mL BSA for 1.5 h at room temperature. Subsequently, the beads were washed three times again in RIPA buffer and half of washed/blocked beads was added per 100 pmol of biotin-DNA oligonucleotides. Then, the mixture was rotated for 4 h at 4 °C. Beads were captured using magnets (Life Technologies) and washed three times with RIPA buffer supplemented with 500 mM NaCl. RNAs and proteins were extracted from the beads and used for further analysis.
RNA immunoprecipitation assay (RIP)
Briefly, the 107 cells were washed with cold 1× PBS three times and irradiated in a UV cross-linker (400 mJ/cm2, 2 min). The whole cells were harvested in ice-cold lysis buffer (10 mM HEPES, pH 7.4, 200 mM NaCl, 30 mM EDTA, 0.5% Triton-X 100, 100 units/mL RNasin Plus RNase Inhibitor (Promega), 1.5 mM DTT, 1× protease-inhibitor cocktail (Roche)) and sonicated for 5 min with an ultrasonic disruptor (SONICS). The cell suspension was centrifuged at 13,000 g for 15 min at 4 °C and the supernatant was collected. A 200-µL sample of the supernatant was saved as input. Subsequently, anti-AGO2 (Proteintech) or IgG (Sangon Biotech) antibody was added into the cell suspension and incubated for 2 h at 4 °C. Protein G Dynabeads (Life Technology) suspension was washed three times with RIPA buffer and then blocked with 500 ng/µL yeast total RNA and 1 mg/mL BSA for 1.5 h at room temperature. The beads were washed three times again in RIP buffer and then added into cell suspension for binding for at least 4 h at 4 °C. The antibody–protein G bead complexes were washed five times with lysis buffer and digested with 30 µg of proteinase K at 65 °C for 1 h. Finally, the immunoprecipitated RNA was purified and detected by quantitative real-time PCR. Antibody validation is provided on the manufacturers’ websites.
Chromatin immunoprecipitation assay (ChIP)
ChIP was carried out as previously described, with modifications [51]. Cells were cross-linked in a UV cross-linker (UVP) at 200-mJ strength. Cell granules were lysed in 1 mL of SDS lysis buffer (1% (w/v) SDS, 10 mM EDTA, and 50 mM Tris-HCl, pH 8.1, Complete protease-inhibitor cocktail (Roche)) after being washed with cold-PBS three times and then were incubated for 20 min on ice. The cell mixture was sonicated for 5 minutes with an ultrasonic disruptor (SONICS) to obtain up to 500-bp DNA fragments. A 100-µL sample of the supernatant was saved as input. The chromatin solution was immunoprecipitated with an antibody to c-MYC (Proteintech, validation provided on the manufacturer’s website) or IgG, and the mixture was rotated for 2 h at room temperature. Then, Protein G Dynabeads (Life Technology) suspension was washed with lysis buffer three times and blocked with 500 ng/µL yeast total RNA and 1 mg/mL BSA for 1.5 h at room temperature. The beads were washed three times again in lysis buffer and then added into cell suspension for binding for at least 4 h at room temperature. The beads were collected and digested with proteinase K for 1 h at 45 °C, and the DNA was extracted by an Endo-free Plasmid DNA Mini Kit (OMEGA). The eluted DNA was subjected to quantitative real-time PCR using the corresponding PCR primers to detect the enriched genomic DNA region.
Dual luciferase reporter assay
Approximately 1 × 104 human U251 cells were co-transfected with 50 nM empty pmirGLO-NC, pmirGLO-LPP-AS2-wt (or pmirGLO-EGFR-wt) or pmirGLO-LPP-AS2-mut (or pmirGLO-EGFR- mut) (RiboBio) and 50 nM miR-7-5p mimics or miR-NC using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocols. The firefly luciferase gene in the vector pmirGLO-control (Promega) was used as the endogenous control to detect transfection efficiency. Firefly and Renilla luciferase activities were measured with a Dual-Luciferase Reporter Assay System (Promega) after 48 h transfection. Firefly luciferase activity was normalized to the corresponding Renilla luciferase activity. Experiments were performed in triplicate, and the data are represented as the mean ± SD.
Cell proliferation assay
Proliferation of U251 and SHG44 glioma cells was measured by using the Cell Counting Kit-8 (Biosharp) assay. A total of 4 × 103 U251 or SHG44 glioma cells/well was seeded in 96-well plates and incubated in a 5% CO2 atmosphere at 37 °C. 10 µL of CCK-8 solution was added to each well at 0, 24, 48 and 72 h after cell transfection. After incubation for 3 hours, the absorbance value per well was determined with an ultraviolet spectrophotometer at 490 nm.
Apoptosis detection by flow cytometry and TUNEL assay
U251 and SHG44 cells (2 × 105 cell/well) were seeded into 6-well plates and harvested at 48 h post-transfection. The cells were washed with PBS, centrifuged twice and resuspended in Annexin-V binding buffer. Annexin V-FITC/PI staining was performed according to the manufacturer's protocols and the apoptosis rate was analyzed by Gallios flow cytometry (BECKMAN COULTER). In addition, cell apoptosis was measured by One-Step TUNEL Apoptosis Kit (RiboBio) according to the manufacturer's introductions.
Cell migration and invasion assay
Cell migration and invasion abilities were detected by Transwell assays. For cell migration detection, transfected U251 or SHG44 cells (1 × 104 cells) were harvested after 24 h transfection and resuspended in 100 µL serum-free medium. Then, the cells were seeded into the upper chamber of a Transwell assay insert (Millipore), and 700 µL 10% FBS medium was added to the lower chamber. After incubation at 37 °C for 48 h, the cells on the lower side were washed three times with PBS, fixed in 4% paraformaldehyde for 20 minutes, and stained with crystal violet solution for 15 minutes. Five random fields were chosen to count stained cells for statistics under an inverted microscope (Olympus) and photographs were taken. For the invasion assay, Transwell chambers were coated with Matrigel for 1 h at 37 °C. The transfected cells (1 × 104 cells) were resuspended in 100 µL serum-free medium and seeded into the upper chamber. Then 700 µL 10% FBS medium was added to the lower chamber. After a 48 h incubation period, the invasive ability was evaluated as mentioned previously for the cell migration assay.
Wound-healing assay
Transfected U251 and SHG44 cells (5 × 104 cells) were seeded into each side of a Culture-Insert 2 Well (Ibidi), and the µ-Dish was filled with 2 mL 2% FBS medium. Images of the different stages of wound healing were photographed via microscopy at 0, 12 and 24 h. Relative wound-healing rates were calculated by using CellSense Standard software (Olympus) and each experiment was performed in triplicate.
Colony formation assay
For colony formation assays, transfected U251 or SHG44 cells were harvested after 24 h transfection and 300 cells were inoculated into 6-well plates. After incubation at 37 °C for 14 days, the colonies were fixed with 4% paraformaldehyde and stained with crystal violet solution.
Immunohistochemistry (IHC)
Immunohistochemistry used a previously described method [52]. Dissected tumors from the mouse model were fixed overnight in formalin solution, dehydrated in ethanol, embedded in paraffin, and cut into 5 µm sections. Then, the specimens were treated with xylene and ethanol to remove paraffin. The slides were blocked with 5% normal goat serum and incubated with anti-Ki67, anti-MMP-9 or anti-EGFR antibodies overnight at 4 °C and washed three times with PBS. After incubation with an HRP-conjugated secondary antibody, the sections were counterstained with hematoxylin. The average integral optical density of each positively stained slide was measured using ImageJ software. Three fields were chosen randomly from each section for measurement.
Western blotting
For western blotting, samples were separated by 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore). The membranes were processed according to the ECL western blotting protocol (GE Healthcare) and scanned with Amersham Imager 680 (GE Healthcare). The following primary antibodies were used: anti-EGFR (BBI, D260292); anti-PI3K (BBI, D155308); anti-AKT (Proteintech, 10176-2-AP); anti-p-PI3K Tyr458 (Cell Signaling Technology, 4228S); anti-p-AKT Ser473 (Proteintech, 66444-1-Ig); anti-c-MYC (Proteintech, 10828-1-AP) and anti-β-actin (BBI, D110001). The anti-β-actin antibody was used as an endogenous control for normalization. Antibody validation is provided on the manufacturers’ websites.
Data deposition
RNA-sequencing data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE153692.
Statistical analysis
All experiments were performed in triplicate; data were analyzed using SPSS (version 23.0) or GraphPad Prism software (version 7.0), and the results are presented as the mean ± SD. Student’s t tests were used to calculate P values, as indicated in the figure legends, while one way analysis of variance (ANOVA) was used for multiple data groups. Correlations between lncRNA LPP-AS2 and miR-7-5p as well as EGFR in human specimens were analyzed by Spearman’s rank test. Survival curves were generated using the Kaplan-Meier method and log-rank tests. P value < 0.05 was considered statistically significant.