A total of 108 FFPE glioma specimens from surgically resected and pathologically confirmed gliomas were collected from 2016.01-2019.12 in our Department of Neurosurgery. All the patients underwent surgery for the first time had not undergone radiotherapy or chemotherapy before surgery. The patients consisted of 73 males and 35 females, with an average age of 59.2 ± 7.3 years. In addition, 34 normal brain tissues were collected from patients undergoing internal decompression for brain injury as a control.
Cell culture and transfection
The human brain normal glioma cell line HEB and the human glioma cell lines U87, U373, A172, U251 and SHG44 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The HEB cell line and U87, U373, A172, and U251 glioma cell lines were cultured in DMEM containing 10% fetal bovine serum. SHG44 cells were cultured with RPMI-1640 medium containing 10% fetal bovine serum. All cells were digested with 0.25% trypsin and incubated at 37 ℃ in 5% CO2 under 95% relative humidity. The cells were digested and sub cultured once every 2–3 days, and cells at the logarithmic growth stage were taken for experiments. The cells were seeded in a six-well plate at a density of 1 × 106/well. When the cell density reached 80%, the cells were transfected with Lipofectamine 2000 (11668-019, Invitrogen, New York, California, USA) according to the manufacturer’s instructions. The medium was changed, and cells were incubated at 37 ℃ in a 5% CO2 cell incubator for 6 h before being collected 36–48 h after transfection. Si-FOXK1, si-NEAT1, miR-128 mimic, mimic NC, si-KDM3A, sh-NC and sh-NEAT1 were chemically synthesized by Shanghai GenePharma.
Using the JASPAR (http://jaspar.genereg.net/) and UCSC (http://genome.ucsc.edu/) websites, possible FOXK1-binding sites in the NEAT1 promoter were identified. According to the sites, the corresponding mutant recombinant luciferase reporter gene vectors and FOXK1 expression vector were co-transfected into U87 and SHG44 cells for verification by dual-luciferase reporter experiments. Si-NC and si-FOXK1 were co-transfected to test whether FOXK1 binds the NEAT1 promoter. After 48 h of transfection, cells were collected and lysed, and the luciferase reporter gene activity was detected by a luciferase detection kit (D0010, Solarbio, Beijing, China) and a dual-luciferase reporter gene analysis system (Promega, Madison, WI, USA).
The wild-type (WT) or mutant (MUT) sequences at the 3'UTRs of NEAT1 and KDM3A were synthesized and individually cloned into the pGL3 reporter vector according to the instructions of the luciferase detection kit. Lipofectamine 2000 reagent was used to co-transfect glioma cells with each wild-type or mutant plasmid and vector containing miR-128 mimic or mimic NC. Forty-eight hours after transfection, luciferase activity was detected by dual-luciferase assay. Each experiment was repeated three times.
Chromatin immunoprecipitation (ChIP) experiment
Cells in logarithmic growth phase were incubated in formaldehyde at a final concentration of 1%. The cells were incubated at 37 ℃ for 10 min to crosslink the cells with formaldehyde. Centrifugation was carried out at 4 ℃ and 2000 × g for 5 min. The supernatant was discarded, and SDS lysis buffer was added before sonication. The ultrasonication conditions were 4.5 s of ultrasonication with 9 s intervals for 14 cycles. The sonicated supernatant was centrifuged at 10,000 × g for 10 min at 4 ℃. The supernatant was divided into two tubes; anti-FOXK1 antibody (ab18196, Abcam, UK) was added to one tube, and negative control rabbit IgG (ab172730, Abcam) was added to the other tube, after which the tubes were incubated at 4 ℃ overnight. The next day, protein A agarose/salmon sperm DNA (cat # 16157, Sigma-Aldrich, USA) was added to each tube to precipitate the immune complexes. The supernatant was obtained by centrifugation at 10,000 × g for 5 min at 4 ℃ and removed. Nonspecific complexes were eluted with an eluent and decrosslinked at 65 ℃ overnight. A gel recovery kit (B110092, Sango Biotechnology, Shanghai, China) was used to purify and recover DNA fragments for qPCR to detect the binding of FOXK1 and NEAT1.
RNA pulldown assay
This experiment was performed as described previously . U87 and SHG44 cells were transfected with biotinylated miR-128, biotinylated miR-128 Mut or biotinylated negative control. After culture for 48 h, the cells were incubated with M-280 streptavidin magnetic beads (11205D, Thermo Fisher Scientific, Waltham, Massachusetts, USA). After incubation at 4 ℃ for 3 h, the cells were washed three times with precooled lysis buffer and once with high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris HCl (pH 8.0) and 500 mM NaCl). The purified RNA was used to detect the level of NEAT1.
RNA-binding protein immunoprecipitation (RIP)
RNA immunoprecipitation (RIP) was performed according to the instructions of an Imprint® RNA Immunoprecipitation Kit (RIP-12RXN, Sigma-Aldrich, St. Louis, Missouri, USA). When the glioma cells reached 80–90% confluence, they were completely lysed with RIP lysis buffer. One hundred microliters of the whole-cell extract were incubated with RIP buffer containing magnetic beads bound to anti-Ago2 antibody (HPA058075, Sigma-Aldrich). The kit contained a negative control antibody, normal mouse IgG antibody. Coprecipitated RNA was isolated using TRIzol reagent (TaKaRa), and RNA levels were detected by reverse transcription PCR.
Glioma cells in logarithmic growth phase were digested and plated in 96-well plates, with approximately 1 × 104 cells per well. Cell transfection was performed the next day, and cells were cultured for 24 h, 48 h, and 72 h. Ten microliters of CCK-8 solution (C0037, Beyotime) was added, and the cells were placed in an incubator for 2 h, after which the absorbance of each well at 450 nm was detected, and a cell growth curve was drawn.
Cell invasion and migration experiments
ECM gel (E1270, Sigma) was thawed in advance at 4 ℃ and moved to an ice box before the experiment began. ECM gel was diluted in serum-free medium at a ratio of 1:8 to form a working solution on ice. The cultured cells were digested, centrifuged and diluted to 2.5 × 105/ml with serum-free medium. Then, the ECM solution was gently pipetted on ice, and 40 µl of the ECM solution was added to the upper chamber of each well in a Transwell apparatus. The ECM solution was incubated at 37 ℃ for 15 min until the gel solidified. Cell suspension (200 µl) was added to the upper chamber of each well, and 500 µl of medium containing 10% FBS was added to the lower chamber. The samples were incubated at 37 ℃ for a few hours, and excess fluid was aspirated from the upper chamber. The cells were washed twice with PBS and fixed with 5% glutaraldehyde at 4 ℃. A 0.1% crystal violet solution was added, and the cells were stained at room temperature for 0.5 h. The cells were washed twice with PBS, following which cells on the upper surface were wiped off with a cotton ball, and the remaining cells were observed under a microscope.
A pen was used to draw a horizontal line evenly every 1 cm underneath a 6-well plate. Approximately 5 × 105 cells were added to each well. After the cells had been seeded, a 200 µl pipette tip was used to make cell scratches perpendicular to the plate. The old medium was aspirated, and the cells were washed 3 times with PBS. After removing the unadhered cells, serum-free medium was added. The cells were incubated at 37 ℃ in a 5% CO2 incubator. Photos were taken at 0 h and 48 h. The images were analyzed with Image-Pro Plus 6.0 (Media Cybernetics, USA). The cell migration distance at each time point was calculated as the cell spacing at 0 h - cell spacing at that time.
Cell cycle and apoptosis
Flow cytometry was used to detect the cell cycle and apoptosis. After 48 hours of transfection, the cells in each group were cultured in the dark for 30 minutes with propidium iodide (PI). Cultures were collected, and the cell cycle was analyzed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA). Data were evaluated as a percentage distribution of cells in G0/G1, S, and G2/M phases. Cultures were performed according to the instructions of an Annexin V-FITC Apoptosis Detection Kit (C1062M, Beyotime), and apoptosis data were analyzed by flow cytometry . Each experiment was repeated three times.
Tumor formation in rats
Forty-eight healthy Sprague Dawley rats aged 4–5 weeks and weighing 90–110 g were purchased from the Guangdong Medical Experimental Animal Center. U87 and SHG44 cells were randomly divided into groups, and the different groups of cells were divided into the sh-NC and sh-NEAT1 groups. Rats were anesthetized intraperitoneally (30 mg/kg sodium pentobarbital) and fixed on a stereotactic apparatus. The intersection of the coronary suture and the sagittal suture was exposed surgically. At this point, a dental drill with a 1 mm bit was used to drill through the skull, the dura mater was punctured with a 10 L1 microthruster, and the needle tip was retracted by 1 mm. A total of 1 × 105 suspended cells (transfected with sh-NC or sh-NEAT1) were injected. Tumor volume was measured by magnetic resonance imaging (MRI) after 2, 3, 4, and 5 weeks of feeding. Rats in each group were sacrificed, and their brain tissue was collected for subsequent qRT-PCR and immunohistochemistry experiments.
The rat brain tissues were fixed in a 4% paraformaldehyde phosphate buffer solution, embedded in paraffin, sectioned, and then baked in a 50 ℃ incubator for 2 h. The sections were dewaxed with xylene, dehydrated in an alcohol gradient for 2 min at each step, and finally placed into distilled water. The sections were boiled in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) for 15–20 min, cooled to room temperature, and washed with PBS. Goat serum blocking solution was added and incubated for 20 min to remove excess liquid. Anti-KDM3A primary antibody (ab106456, Abcam) was added and incubated overnight at 4 ℃. After washing with PBS, goat anti-rabbit IgG (ab205718, Abcam) secondary antibody was added, and the cells were incubated at room temperature for 1 h. The cells were washed 3 times with PBS for 3 min each. The signal was developed by incubation with DAB for 5–10 min, following which the cells were redyed with hematoxylin for 2 min, and observed under a microscope after routine dehydration, clearing, and mounting.
TRIzol was used to extract total RNA from tissues and cells. 5 µg RNA was used for reverse synthesis to generate cDNA with a Superscript III RT Reverse Transcription Kit (#11752050, ABI Invitrogen). The TaqMan microRNA assay (Thermo Fisher Science) with cDNA as the template was used for qRT-PCR. The reaction was carried out as follows: 95 ℃ for 2 min, followed by 45 cycles at 95 ℃ for 15 s and 60 ℃ for 45 s. U6 was used as an internal parameter to normalize the results.
qRT-PCR to quantify mRNA was carried out according to the instructions of the TaqMan gene expression assay (Applied Biosystems, Foster City, CA, USA). GAPDH was used as an internal reference. The PCR program was designed as follows: 95 ℃ for 2 min, followed by 40 cycles at 94 ℃ for 20 s, 60 ℃ for 20 s, and 72 ℃ for 30 s. The primer sequences are shown in Table 1. The relative transcription level of each target gene was calculated by the relative quantitative method (2-△△CT method) .
Total protein was extracted from tissues or cells, and the protein concentration was measured using a BCA kit (Thermo, USA). Polyacrylamide gel electrophoresis of samples containing 30 µg of protein was performed at a constant voltage of 90 V for 30 min and 120 V for 50 min. After electrophoresis was completed, proteins were transferred to a PVDF membrane, and the membrane was blocked with 5% skim milk powder at room temperature for 1 h. The membrane was then incubated with the following antibodies at 4 °C overnight: rabbit anti-FOXK1 polyclonal antibody (ab18196, 1:1000, Abcam), rabbit anti-KDM3A monoclonal antibody (ab106456, 1:100, Abcam), rabbit anti-β-catenin polyclonal antibody (ab16051, 1:500, Abcam), rabbit anti-c-Myc monoclonal antibody (ab32072, 1:1000, Abcam), rabbit anti-cyclinD1 monoclonal antibody (ab16663, 1:200, Abcam), and rabbit anti-β-actin monoclonal antibody (ab179467, 1:5000, Abcam). The membrane was washed 3 times with PBST (PBS buffer containing 0.1% Tween-20). Then, horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (ab205718, 1: 2000, Abcam) was added and incubated with the membrane at room temperature for 1 h. The membrane was washed 3 times with PBST buffer. After scanning and development with an optical luminometer (GE, USA), the protein bands were scanned using Image-Pro Plus 6.0 software for grayscale analysis of relative protein expression.
The data in this study were analyzed using SPSS 21.0 statistical software (SPSS, Inc., Chicago, IL, USA). Counted data are represented by the number of cases and were analyzed by the chi-square test. Measured data are expressed as the mean ± standard deviation. Comparisons between two groups were performed using the t-test, comparisons between multiple groups were performed using single-factor analysis of variance, and post hoc tests were performed using Tukey’s test. P < 0.05 indicates that a difference was statistically significant.