Sample collection
Tissue specimens of glioma: From January 2010 to June 2019, a total of 60 pathologically confirmed primary glioma specimens were collected from the First Affiliated Hospital of USTC according to the WHO (2000) gliomas grading standard, Ⅰ/Ⅱ/Ⅲ/Ⅳ grade glioma, 15 patients with craniocerebral trauma afterbrain swelling induced by cerebral herniation after cranial decompression surgery for 12 cases of normal brain tissue specimens, as normal controls. All specimens were instantly frozen after collection in liquid nitrogen, then transferred to the -80℃ refrigerator for storage. Prior to collection, the above specimens have gained approval from the ethics Committee of the First Affiliated Hospital of USTC and written consent of the patients and their families has been obtained.
Cell culture
Normal brain tissue cells HEB and glioma cells HS683, T98MG, U373, A172, SHG-44, U87 and U251 were all obtained in the cell bank of The Chinese Academy of Sciences, then were cultured in DMEM high glucose medium of 10% fetal bovine serum, at 37°C and 5% CO2 in an incubator.
Quantitative real-time PCR (qRT-PCR)
We extracted total RNA from glioma tissues and cells with Trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA). We adopted a Nanodrop spectrophotometer (ND-100) to detect the concentration of RNA and at the 260/280 nm ratio detect its quality. Via a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA) we generated the complementary DNA (cDNA). We adopted Two-Step SYBR PrimeScript RT-PCR Kit (Takara Bio, Inc, Japan) to perform qRT-PCR (quantitative real-time PCR) for the assays of LINC00657, PUM2 and NANOS3. RT-qPCR, on a Roche Light Cycler480 system (Roche Diagnositcs, Inc.), in conformity with the manufacturer’s instructions. We normalized expressions to endogenous controls (GAPDH), also we used the relative quantification (2−DDCt) method to perform fold change calculation. The sequences of the primers were as follows:
LINC00657 F, 5’-CAGAGGAGGTATGCAGGGAG-3’ and R, 5’-GGATGTCTAGCTCCAAGGGG-3’;
NANOS3 F, 5’-CATTTATTGAGGGCTGACTGGAT-3’ and R, 5’-CGGAACTCCTGTGCTTTGTCT-3’;
PUM2 F, 5’-TCGGGGAATGGGAGAGCTTT-3’ and R, 5’-GCTGGGACATTGAATGGTGAGA-3’;
GAPDH F, 5’-TCAAGATCATCAGCAATGCC-3’ and R, 5’-CGATACCAAAGTTGTCATGGA-3’;
Lentiviral Vector Construction and Infection
Respectively, we ligated short hairpin RNAs directed against LINC00657 and NANOS3 into the pLV-u6-gfp-Puro lentiviral vector (Genechem, Shanghai, China). And we ligated short hairpin RNA directed against PUM2 into the pLV-u6-red-bsd lentiviral vector (Genechem, Shanghai, China). We also ligated the NANOS3 coding sequence (CDS) into the pLV-cmv-red-bsd lentiviral vector (Genechem, Shanghai, China). We also ligated the PUM2 coding sequence (CDS) into the pLV-cmv-gfp-Puro lentiviral vector (Genechem, Shanghai, China). We harvested lentivirus 48 h after that the packaging vectors co-transfected with the lentiviral vectors or the empty lentiviral vectors (negative control, NC) into U87 and U251 cells. We consequently infected cells with the lentivirus, so as to obtain the sh-LINC00657, sh-PUM2 and sh-NANOS3 cells, or PUM2 and NANOS3 overexpressing cells.
Cell Proliferation Assay
To ananlyze the proliferation of U251 and U87 cells, we performed Cell Counting Kit-8 assays (CCK-8, Dojin, Japan). In 96-well plates we seeded cells in a density of 2,000 cells per well after the transfection. We added 10 μl of CCK-8 solution into every well after 72 h, then at 37℃ we incubated the cells for 2 h. With the SpectraMax M5 microplate reader, at 450 nm we measured the absorbance.
Quantization of Apoptosis by Flow Cytometry
We quantified cell apoptosis via the Annexin 7AAD/PE staining (Southern Biotech, Birmingham the AL, USA). The cells, which were rinsed with PBS for twice and centrifugalized, suffered being stained with Annexin 7AAD/PE and being resuspended in Annexin-V-7AAD/PE binding buffer, in obedience to the manufacturer’s instructions. To get the apoptotic fractions, we then used flow cytometry (FACScan, BD Biosciences) to analyze the cells.
Cell Migration Assay
To test the invasion and migration of U251 and U87 we adopted Chambers (24-well) with 8-μm pore size (Costar, Corning, NY, USA). Processes were performed in a manner that the cells suffered being resuspended at a density of 105/ml in 100 μl serum-free medium, also being seeded in the upper chamber [or chambers precoated with 500 ng/ml Matrigel solution (BD, Franklin Lakes, NJ)]. In the lower chamber, we placed six hundred microliters medium of 10% FBS for 48 h; with a cotton swab we then wiped the cells on the upper membrane surface out physically. With methanol and being stained with 10% Giemsa, cells which were migrated to the lower side of the membrane were finally fixed. Then, under a microscope we chose five random fields for counting the cells, and took the photographs.
Western Blot Assay
We extracted the total protein from frozen cells with RIPA (radioimmunoprecipitation assay) buffer containing 50mM HEPES, 1mM EDTA (ethylenediaminetetraacetic acid) (pH 8.0) on ice. We centrifuged the samples were at 17,000 rpm and 4℃ for 40min, and by BCA (bicinchoninic acid) protein assay kit (Beyotime, Shanghai, China) we obtained the protein concentration of the supernatant extracts. After that, they were subjected to SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and then transferred to PVDF (polyvinylidene fluoride) membranes electrophoretically. We incubated such membranes in Tris-buffered saline which contained 5% nonfat milk at 25℃ for 2 h and then incubated them with primary antibodies as follows: PUM2 (1:2,000, ab92390, Abcam, Cambridge, MA, USA), NANOS3 (1:2,000, ab70001, Abcam, Cambridge, MA, USA) and GAPDH (1:1,000, ab8245, Abcam, Cambridge, MA, USA) for 18 h. As secondary antibodies, we used Horseradish peroxidase (HRP)-linked antimouse inmmunoglobulin G (IgG) and HRP-linked antirabbit IgG antibodies. were Via FluorChem 2.0 software (Alpha Innotech, San Leandro, CA, USA) we quantified the signals.
RNA Immunoprecipitation Assay
Via a Magna RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) we performed RNA immunoprecipitation (RIP) We incubated whole-cell lysate with RIP buffer containing magnetic beads, which were conjugated with negative control normal rabbit IgG or with human anti-PUM2 antibody. We incubated samples with Proteinase K and isolated the immunoprecipitated RNA. By a spectrophotometer (NanoDrop, Thermo, Scientific, Waltham, MA, USA) we measured the RNA concentration, and via a bioanalyzer (Agilent, Santa Clara, CA, USA) we assessed the RNA quality. Besides, we extracted purified RNAs and analyzed them to elucidate the presence of the binding targets.
RNA Pull-Down Assay
Via Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) we examined the interaction betwixt LINC00657 and PUM2. We coincubated biotin-labeled LINC00657 with magnetic beads and protein extract of U251 or U87 cells. For generating the bead–RNA–protein complex we used low speed centrifugation. Washed with Handee spin columns, in SDS buffer the bead compound was boiled, and via the GAPDH control we detected the retrieved protein.
Tumor Xenografts in Nude Mice
We purchased female nude mice from GemPharmatech (Nanjing, China) at 6-8 weeks. This experimental study meets the requirements of animal ethics, minimizing the number of animals used, and reducing the pain of animals under anaesthesia before operation. During the experiment, the room temperature was controlled at about 22℃ and the relative humidity was about 60%. During the study we gave autoclaved food and water to the animals received. Into 3 groups the nude mice were divided: NC group, sh-LINC00657 group and sh-LINC00657+PUM2 group. We used the stable-expression U87 cells for in vivo study. We injected 100l cells into the right side of the mouse near armpit. We measured he tumor size with vernier caliper every 4 days after the formation of the transplanted tumor and recorded. We calculated the tumor volume with this formula: volume (mm3) = length × width2/2.
Statistical Analysis
We presented data as mean ± SD. By SPSS 18.0 statistical software we evaluated all statistical analyses, via the Student’s t test or one-way analysis of variance (ANOVA). When P < 0.05 differences are recognized to be influential.