Human tissue samples
Messenger RNA (mRNA) expression data for 158 glioma samples were downloaded from the Chinese Glioma Genome Atlas (CGGA) data portal (http://www.cgga.org.cn/portal.phpg). The 158 glioma samples included 48 astrocytomas (As), 13 oligodendrogliomas (Os), 8 anaplastic astrocytomas (AAs), 10 anaplastic oligodendrogliomas (AOs), 15 anaplastic oligoastrocytomas (AOAs) and 64 glioblastoma multiforme tumours (GBMs). Tissue samples were obtained from the Department of Neurosurgery in Xuzhou Central Hospital from 2012 to 2015. Of these samples, three were normal brain tissues (NBTs), and twelve (3 grade II, 4 grade III and 5 grade IV) were glioma samples. The NBT samples were obtained from three patients who suffered severe brain trauma.This study was approved by the Medical Review Board of Xuzhou Central Hospital.
The U251 cell line was purchased from the Chinese Academy of Sciences Cell Bank and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml) (all from Invitrogen, Carlsbad, CA, USA) at 37 °C under a humidified atmosphere of 5% CO2.The U251 cell line was are authorized by the Chinese Academy of Sciences Cell Bank and Xuzhou Central Hospital for usement.
Reagents and transfection
The recombinant plasmid for the pcDNA3.1 vector, which contains the ORF of human AdipoR2, was chemically synthesized and purified by GeneChem (Shanghai, China). The empty pcDNA3.1 vector was used as a negative control (NC). All plasmids were transfected into cells using Lipofectamine 2000 Transfection Reagent (Invitrogen, USA) according to the manufacturer’s instructions. The selective AMPK inhibitor compound C (iAMPK) was purchased from Calbiochem (La Jolla, CA).
RNA was extracted from tissues using TRIzol Reagent (Invitrogen). AdipoR1 and AdipoR2 qRT-PCR reactions were performed using Fermentas reverse transcription reagents and SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s protocols. GAPDH was used for normalisation. Relative gene expression was calculated via the 2-△Ct method.
WST-8 cell growth assay
U251 cells were seeded in 96-well culture plates at 2000 cells/well/100 µL. The cells were treated with AdipoR2 for 1–4 days. Then, tetrazolium monosodium salt WST-8 (Dojindo, Japan) was added (10 µL/well). After incubation for 2 h, the absorbance was determined using a microplate reader (Bio-Rad, USA) at a wavelength of 450 nm with the reference wavelength set at 630 nm.
Colony formation assay
U251 cells were seeded in six-well plates and cultured overnight. Then, AdipoR2 or NC was transfected into the cells. After 48 h, 5×102 treated and untreated cells were independently plated onto 60 mm tissue culture plates. After incubation for 2 weeks, visible colonies were fixed with 4% methanol for 30 min and stained with 0.1% crystal violet for 20 min. Colony-forming efficiency was calculated using the following equation: Colony-forming efficiency = number of colonies/number of plated cells×100%.
Cell cycle assay
Forty-eight hours posttransfection, U251 cells were collected and fixed with 70% ethanol at -20°C overnight. DNA was stained by incubating cells in 50 mg/mL propidium iodide (PI) (Sigma-Aldrich, USA) and 10 mg/mL RNase A (Boehringer-Mannheim, Germany) for 1 h at room temperature. The cells were then analysed by FACScan (Becton-Dickinson, USA).
Proteins were extracted in lysis buffer according to the manufacturer’s protocol. Lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, USA). The membranes were incubated in blocking buffer.The membranes were incubated at 4°C overnight with primary antibodies against the following proteins:AdipoR2(Abcam,USA),AMPK(Abcam,USA),p-AMPK(Abcam,USA),mTOR(Abcam,USA),p-mTOR(Abcam,USA),S6K(Abcam,USA), pS6K(Abcam,USA), S6P(Abcam,USA)), phosphorylated (Ser240/244) S6 ribosomal protein (pS6P) and GAPDH (CST, USA). Immunoreactivity was visualized with horseradish peroxidase-conjugated goat anti-rabbit antibody (Bioworld, USA). The protein bands were detected and imaged with a ChemiDocXRS+ Gel Imaging System (Bio-Rad, USA) and analysed by densitometric quantification using ImageJ software.
Gene set enrichment analysis with AdipoR2 expression
The gene expression profiles of glioma samples from CGGA were analysed by GSEA15. Pearson's correlation was used to analyse the relationship between AdipoR2 and all identified genes with MATLAB software (P<0.001). GSEA (http://www.broadinstitute.org/gsea/) analysis was used to identify pathway gene sets that were correlated with the AdipoR2 expression profile. For GSEA, AdipoR2 expression was treated as a binary variable divided into low or high AdipoR2 expression, and the cut-off point was 50%. As a metric for ranking genes in the GSEA, the difference between the means of samples with low and high AdipoR2 expression was used, and the other parameters were set to their default values.
Kaplan-Meier survival analysis was used to estimate survival distributions. The log-rank test was used to assess the statistical significance between stratified survival groups using GraphPad Prism. The t test was used to determine differences in each 2-group comparison. All statistical analyses were performed using MATLAB 2012, SPSS software for Windows, or GraphPad Prism (GraphPad Software). All data are presented as the means ± standard error. A 2-sided P value of < 0.05 was regarded as significant.