Cell Culture and Reagents
The human glioma cell lines of U251 and A172 cells were purchased from the American Type Culture Collection (ATCC, Manassas, USA) and the Chinese Academy of Sciences Cell Bank (Shanghai, China), respectively. The U251 and A172 cells were cultured in Dulbecco’s Modified Eagle’s Medium(DMEM, Gibco, Rockville, MD, USA) containing 10% fetal bovine serum( FBS, Gemini, California, USA), supplemented with 2 mM glutamine (HyClone, Logan, Utah, USA) and 100 U/ml penicillin–streptomycin (HyClone, Logan, Utah, USA) at 37 oC with 5% CO2. The Temozolomide (TMZ) and LiCl were purchased from the Sigma Chemical Co., Ltd. (Sigma, Loui- siana, USA).
Cell Transient Transfection
The overexpression plasmid pCDH-MSCV-MCS-EF1-GFP-PU (pMSCV) was generously donated by Dr. Mo at the Cancer Institute of the University of Mississippi Medical Center. As for the construct of pcDNA3.1-lncRNA GAS5 plasmid, the whole sequence of GAS5 was synthetized and subcloned into pcDNA3.1(+) plasmid (pMSCV-GAS5) by Shanghai Novoprotein Technology Co., Ltd. (Shanghai, China). The small interfering RNA (siRNA) sequences were designed by Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences of siRNAs were shown in Supplemental Table 1. All transfections were completed by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s manuals. The efficiency of transient transfection is detected by quantitative real-time fuorescence polymerase chain reaction (qRT-PCR, Supplemental Fig.S1A ).
Cell Stable Transfection
As for the cell stable transfection, U251 cells were firstly transfected with pMSCV and pMSCV-GAS5. Then the transfected cells were seeded in twelve-well culture plates at a confuency of less than 25% to stably select for at least 7 days with puromycin dihydrochloride (Beyotime, Shanghai, China) at the concentration of 2 μg/ml. The stably transfected pMSCV-GAS5 (sMSCV-GAS5) and pMSCV (sMSCV) U251 cells were ultimately harvested. The efficiency of stable transfection was confrmed by qRT-PCR (Supplemental Fig.S1B).
RNA Extraction, Reverse Transcription and qRT‐PCR analysis
The total RNA was extracted from cell lines using RNAeasy™ Animal RNA Isolation Kit with Spin Column (Beyotime, Shanghai, China), according to the manufacturer’s protocols. Then extracted RNAs were reversed to complementary DNAs(cDNAs) through using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Kusatsu, Japan). Subsequently, the qRT‐PCR detection of mRNA was performed using the TB GreenTM Premix Ex Taq™ II (Tli RNaseH Plus) kit (Takara, Kusatsu, Japan) on the CFX ConnectTM Real-Time System (Bio-Rad, Singapore). The special primers were synthesized from TSINGKE Biological Technology Co., Ltd. (Chongqing, China). The relative expression levels of RNAs were normalized to GAPDH and calculated by the method of 2− ΔΔCt. The primer sequences were provided in Supplemental Table 2.
Flow Cytometry Apoptosis Assay
According to our pervious study, the half maximal inhibitory concentrations (IC50s) of TMZ in A172 and U251 cells were 1200 µM and 150 µM, respectively. Based on this result, the transfected U251 and A172 cells were treated with TMZ at IC50 doses for another 72h. The cells were then resuspended in binding buffer containing FITC-Annexin V and propidium iodide (PI) for 15min at room temperature. After diluted with binding buffer, stained cells were immediately analyzed by flow cytometry (Beckman, USA) for determination of the apoptosis rate.
Colony Formation Assay
The stable transfected U251 cells (totally 500) were seeded in six-well plate. After treated with TMZ at IC50 doses for 3 days, the U251 cells were preserved in 10% DMEM containing 10% FBS to form colonies. A week later, the cells were fixed with 4% paraformaldehyde for 15 min and then stained with 0.1% crystal violet for 30min (Sigma, San Francisco, CA, USA). The appeared colonies were captured under microscope and subsequently counted by ImageJ.
Cell Counting Kit‑8 (CCK‑8) Assay
The cell viability was evaluated through Enhanced Cell Counting Kit-8 assay (CCK-8, Biosharp, Beijing, China). Briefly, equal numbers of transfected cells were seeded into 96-well plates (3000/well) and cultured in 10%DMEM overnight. Next, the cells in plates were treated with dose TMZ concentrations for 72h. After indicated transfection and treatment times, cells were treated with CCK-8 solution(10 μl/well) for 1h at 37 oC. The optical desity was read at a wavelength of 450nm using Spectrophotometer Multiskan GO (Thermo Fisher, Vantaa, Finland).
The cells were lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with the protease inhibitor PMSF (Beyotime, Shanghai, China). Subsequently, protein concentration was measured with the BCA Kit (Beyotime, Shanghai, China). Then protein extracts were segregated by 10% sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes ((Millipore, Carrigtwohill, Ireland). After blocking with 5% BSA in Tris-buffered saline with 0.1% Tween 20(TBST) for 1h at 37°C, the membranes were incubated with the primary anti-GAPDH, anti-β-catenin, anti-c-MYC, anti-cyclinD1, anti-PTBP1(all purchased from Proteintech, Rosemont, USA) at 4 °C overnight. Then the membranes were incubated with HRP-conjugated AfniPure goat anti-mouse/ rabbit IgG (H+L) secondary antibody (Proteintech, Rosemont, USA). The immunoreactive bands for target protein were imaged using WesternBright™ ECL reagent (Advansta, Menlo Park, USA) and the band intensities were normalized to those of GAPDH.
For immunofluorescence, the transfected cells were seeded into 12-well plate, which were placed with glass coverslips in advance. After cultured with 10%DMEM overnight, the cells were fixed with 4% paraformaldehyde for 20min and subsequently washed with cold 0.01M PBS for 3 times. Next, the cells were blocked with 5%BSA for 1h at 37 oC and incubated with primary antibodies against PTBP1/β-catenin (Proteintech, Rosemont, USA) at 4 oC overnight. Cells were then labeled with the fuorescent secondary antibody (FITC AfniPure goat anti-rabbit IgG (H+L) 1:100 [EarthOx, San Francisco, USA], Rhodamine (TRITC)- conjugated goat anti-rabbit IgG (H +L) 1:100 [Proteintech, Rosemont, USA]). Cell nuclei were counterstained with DAPI for 5min. Images were captured with the microscope using 40x objectives.
RNA Immunoprecipitation (RIP)
According to the manufacturer’s instructions, the EZ-Magna RIP Kit (Millipore, Carrigtwohill, Ireland) was utilized for performing RIP. The mouse monoclonal anti-PTBP1 were obtained from Millipore (Carrigtwohill, Ireland). Normal mouse anti-IgG (Millipore) were served as the negative control. qRT-PCR was conducted to examined the purified RNA isolated from RIP.
Nude Mice Xenograft Experiments
Male BALB/C nude mice (4–6 weeks old, 18-20 g, SPF, male) were commercially purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China) and maintained in an SPF‐grade pathogen‐free animal laboratory. The xenograft assay was performed as described previously using U251 cells (1 × 108), which stably overexpressed p-MSCV and p-MSCV-GAS5. The mice were randomly divided into two groups (n=6 each group) named s-MSCV and s-GAS5, respectively. About 30 days after xenotransplantation, the mice were injected with TMZ(50 mg/kg/d) dissolved in 5% DMSO PBS solution every 5 days with every five-day interval for two cycles, and the volume of tumors was measured simultaneously. After 45 days, the mice were sacrificed. The tumors were removed to measure the final volumes and weights. The volume was calculated using the formula: volume= (length×width2)/2. All procedures of animal experiment were approved by the Animal Research Ethics Committee of Chongqing Medical University.
In the whole researches, the quantitative data were presented as mean ± SEM. Differences between two groups were analysed by an unpaired t-test. The log-rank test was utilized to evaluate the statistical correlation of survival rate with GAS5 expression. The experimental data, which was at least three independent replicates were analyzed with Graph Pad Prism Version 5.01 (GraphPad, La Jolla, CA, USA). Statistical significance was expressed as *P < 0.05, **P < 0.01, or ***P < 0.001.