Collection of GBM samples
Thirty GBM samples were collected during surgery, including 20 primary GBM cases and 10 recurrent cases, and they were pathologically confirmed. In addition, 5 normal brain samples were collected during decompression surgery of traumatic brain injury at Neurosurgery Department, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China. This study was reviewed and approved by the Clinical Research Ethic Committee of the First Affiliated Hospital of Nanjing Medical University.
Bioinformatic analyses
RNA-seq data were downloaded from The Cancer Genome Atlas (TCGA, http://cancergenome), Chinese Glioma Genome Atlas (CGGA, www.cgga.org) and Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo) for bioinformatic analyses. GO and KEGG were performed using DAVID (https://david.ncifcrf.gov/). The limma R package and clusterprofiler package were used for differential analysis and GSEA analysis, respectively.
Cell culture
Human glioma cell lines (U87, LN229, A172, T98G, U251), normal human astrocytes (NHA) and human umbilical vein endothelial cells (HUVECs) were provided by the American Type Culture Collection (ATCC). Primary GBM cell line (N3) was gifted from Beijing Tiantan Hospital. HEK293T cell line was provided by Cell Bank of the Chinese Academy of Sciences. Except for HUVECs cultured in EGM-2 (Lonza), the remaining were cultivated in DMEM containing 10% FBS. Cells were incubated at 37℃ in a humidified environment containing 5% CO2.
qRT-PCR
Cells were lysed in TRIzol (Invitrogen, CA, USA) and the isolated RNA was reversely transcribed to cDNA using the PrimeScript RT (Takara, Nanjing, China). PARISTM Nuclear/Cytosol Fractionation Kit was used for isolating nuclear and cytoplasmic components. After preparing a PCR system using the SYBR Green Premix Ex Taq (Takara, Nanjing, China), PCR was conducted with U6 or GAPDH as the internal reference. Relative level was calculated using 2-ΔΔCt method. Primer sequences are shown in Additional file 2: Table s4.
Fluorescence in situ hybridization (FISH)
RNA-FISH was conducted as previously described [22]. LBX2-AS1 probe was provided by RiboBio (Guangzhou, China). FISH-RNA signal in GBM cells and specimens were captured using a confocal microscope system (Zeiss LSM 700).
Cell transfection
Transfection of siRNAs, miRNA mimics and plasmids (Genechem, Shanghai, China) was conducted using Lipofectamine 2000 (Invitrogen). cDNAs that were complementary paired to LBX2-AS1, Sp1 and LIF were synthesized, which were cloned into pcDNA3.1 (Invitrogen). LBX2-AS1 shRNAs and negative control (sh-Ctrl) were synthesized by Genechem (Shanghai, China). Screening by puromycin at 48 h, stably expressed N3 and U87 cell lines were established. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) sequences designed for specific targets are listed in Additional file 1: Table s1-3.
Western blot
Western blot was conducted as previously described [23]. The following primary antibodies were used: anti-LIF (Abcam, ab138002), anti-p-STAT3(Abcam, ab76315), anti-STAT3(Cell Signaling Technology, 12640), anti-Sp1(Cell Signaling Technology, 9389), anti-N-cadherin (Cell Signaling Technology, 13116), anti-E-cadherin (Cell Signaling Technology, 3195), anti-Vimentin (Cell Signaling Technology, 5741), anti-Snail (Cell Signaling Technology, 3879), anti-Slug (Cell Signaling Technology, 9585), anti-MMP9 (Abcam, ab76003), anti-VEGF (Abcam, ab69479), and anti-GAPDH (Cell Signaling Technology, 5174).
Cell proliferation assay
Proliferative potential of GBM cells was assessed by CCK-8, colony formation and EdU assay. In the first experiment, cells were seeded in a 96-well plate and absorbance at 450 nm was measured using Cell Counting Kit-8 (Dojindo, Shanghai, China). In colony formation assay, cells were seeded in a 6-well plate and cultivated for 14 days. Colonies were washed in PBS twice, fixed in 4% paraformaldehyde for 10 min and stained in 0.1% crystal violet for 30 min, which were captured under a microscope. EdU Apollo DNA in vitro kit (RiboBio, Guangzhou, China) was used for measuring DNA synthesis during the proliferative process of GBM cells. Cells seeded in a 6-well plate were incubated with 50 µM EdU for 2 h, followed by 30-min fixation in 4% paraformaldehyde and staining with Apollo and Hoechst 33342. EdU-positive cells were captured using a fluorescence microscope for counting.
Transwell assay
Transwell inserts (Corning, New York, USA) were pre-coated with 20 μg/μl Matrigel (BD Biosciences, New jersey, USA). 2×104 cells suspended in serum-free medium and culture medium containing 10% FBS were respectively applied at the top and bottom of the prepared insert. After 24-h cell culture, cells invaded from the top to the bottom were fixed in 4% paraformaldehyde for 10 min and dyed in 0.1% crystal violet for 30 min. Invasive cells in 3 random fields per sample were captured for counting.
3D tumor spheroid invasion assay
3D tumor spheroid invasion assay was conducted as previously described [24]. 2×105 cells in a 96-well plate were cultivated in complete medium containing spheroid formation matrix for 96 h, followed by adding Matrigel for embedding spheroids. Spheroids were captured at 0, 24, 48 and 72 h, respectively under a microscope, and the invasive area was evaluated using spheroids at 0 h as the reference.
Tube formation assay
1×104 HUVECs cultivated in conditioned medium of GBM cells were collected and seeded on a μ-Slide Angiogenesis coated with 10 μl Matrigel (BD Bioscience, New Jersey, NJ, USA) at 37℃ for 30 min. After 3-h cell culture, tube formation was captured under an optical microscope.
Dual-luciferase reporter assay
Promoter-containing vector for LBX2-AS1, Sp1, TEAD2 and KLF5 (Genechem, Shanghai, China) were co-transfected into HEK293T cells, respectively. P1-wt (wild-type) and P1-mut (mutant) sequences were synthesized and cloned into pGL3-basic luciferase vectors (Promega, Madison, USA), which were co-transfected into HEK293T cells with Sp1 plasmid. In addition, luciferase vectors containing mutant or wild-type sequences in which miR-491-5p bound to promoter region of LBX2-AS1 or LIF were co-transfected into N3 or U87 cells with miR-491-5p mimic or negative control. Relative luciferase activity was measured using the Promega Dual-luciferase Reporter System, and normalized to that of Renilla luciferase activity.
Chromatin immunoprecipitation (ChIP)
EZ-ChIP Kit (Millipore, Billerica, MA, USA) was used for ChIP assay. Briefly, cells were cross-linked in 1% formaldehyde for 10 min and terminated by glycine. Cells were lysed to chromatin fragments by sonication. DNA/protein complex was incubated with 3 μg anti-Sp1 (Cell Signaling Technology, 9389). Anti-IgG (Millipore, 12–371) was used as the negative control. The special primers for P1 site, P2 site and P3 site are listed in Additional file 2: Table s5.
RNA immunoprecipitation (RIP)
Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) was used for RIP assay. Briefly, cell lysate was incubated with anti-Ago2(Abcam, ab32381), and anti-IgG (negatively control). A protein-RNA complex was captured, followed by removal of the protein. The magnetic beads were repeatedly washed with RIP washing buffer, and the immunoprecipitated RNA was quantified by performing qRT-PCR.
Immunohistochemistry (IHC)
After sacrifice, GBM tissues collected from nude mice were fixed in 4% paraformaldehyde, and paraffin embedded for preparation of tissue sections. Sections were incubated with anti-Ki-67, anti-LIF and anti-p-STAT3 for IHC.
Orthotopic GBM xenograft model
6-week-old male BALB/c nude mice were provided by Animal Core Facility of Nanjing Medical University. Twelve mice were used in the subcutaneous xenograft GBM model, with 6 mice in each group. They were respectively subcutaneously administrated with 1×107 U87 cells transfected with sh-NC or sh-LBX2-AS1#1. The growth of tumor was measured once a week and the volume was calculated: Tumor volume (mm3) = 0.5 × the longest diameter (mm) × the shortest diameter2 (mm2). In the intracranial xenograft GBM model, 12 mice were randomly assigned to two groups, with 6 in each. They were respectively intracranially administrated with 2.5×105 U87 cells transfected with sh-NC or sh-LBX2-AS1#1 using a stereotaxis instrument. Tumors in mice was examined using optical imaging (IVIS spectrum, PerkinElmer, USA).
Statistical analysis
Statistical analyses were conducted using GraphPad software version 8.0 (GraphPad software, San Diego, CA, USA) or SPSS Statistics 23.0 (SPSS, Chicago, IL, USA). Differences between groups were compared by Student’s t test or one-way ANOVA. Pearson’s correlation test was performed for evaluating the correlation between two indexes. Kaplan-Meier survival analysis was performed, and the difference was compared by log-rank test. All experiments were repeated in triplicate, and data were expressed as mean ± standard error of the mean (SEM). A significant difference was considered at p<0.05 (*p<0.05, **p<0.01, ***p<0.001).