Human tissue samples
Messenger RNA (mRNA) expression data for 158glioma samples was downloaded from the Chinese Glioma GenomeAtlas (CGGA) data portal (http://www.cgga.org.cn/portal.phpg).In total, 158 glioma samples included 48astrocytomas (As), 13oligodendrogliomas (Os), 8 anaplastic astrocytomas (AAs), 10 anaplastic oligodendrogliomas (AOs), 15 anaplastic oli-goastrocytomas (AOAs) and 64 tumors of glioblastoma multiforme (GBMs).Tissue sampleswere obtained from the Department of Neurosurgery in Xuzhou Central Hospital from 2012 to 2015. Of thesetissuesamples, three were normalbrain tissues (NBTs) and twelve(3 grade II, 4 grade III and 5 grade IV) were glioma samples. NBT samples were obtained from three patients who suffered severe brain trauma. This study was approved by the Medical ReviewBoard of Xuzhou Central Hospital.
U251 cell line was purchased from Chinese Academy of Sciences Cell Bank and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 µg/ml) (all from Invitrogen, Carlsbad, USA) at 37 ˚C under a humidified atmosphere of 5% CO2.
Reagents and transfection
The recombinant plasmid for pcDNA3.1 vector, which contains ORF of human AdipoR2 was chemically synthesized and purified by Genechem (Shanghai, China). The blank vector pcDNA3.1 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.Selective AMPK inhibitorcompound C (iAMPK) was purchased fromCalbiochem (La Jolla, CA).
RNA was extracted from tissues using TRIzol(Invitrogen). AdipoR1 and AdipoR2 (qRT-PCR) reactions were performedusing Fermentas reverse transcription reagents andSYBR Green PCR Master Mix (Applied Biosystems)according to manufacturer’s protocols. GAPDH wasused for normalisation. Relativegene expression was calculated via 2−△Ct method.
WST-8 growth assay
U251 cells were seeded in 96-well culture plates at 2000 cells/well/100 µL. 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 450 nm wavelength with the reference wavelength set at 630 nm.
Colony formation assay
U251 cells were seeded in six-well plates and cultured overnight. And thensubsequently, AdipoR2 or NCwas transfected into 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 the stained with 0.1% crystal violet for 20 min. Colony-forming efficiency was calculated as the number of colonies/plated cells × 100%.
Cell cycle assay
After 48 h post-transfection, 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 analyzed by FACScan(Becton- Dickinson, USA).
Proteins were extracted inlysis bufferaccording to the manufacturer’s protocol. Lysates were separated by SDS-PAGEand transferred to anitrocellulose membrane (Bio-Rad, USA).The membranes were incubated in blocking buffer.Themembranes were incubated with the AdipoR2 (Abcam, USA), AMPK, phosphorylated (Thr172) AMPK (p-AMPK), mTOR, phosphorylated (Ser2448) mTOR (p-mTOR),70-kDa ribosomal protein S6 kinase(S6K), phosphorylated(Thr421/Ser424) p70S6 kinase (pS6K), and S6 ribosomal protein (S6P), phosphorylated(Ser240/244) S6 ribosomal protein (pS6P)and GAPDH (CST, USA) primary antibodyat 4 °Covernight, respectively. Immunoreactivity was visualizedwith horseradish peroxidase-conjugated goat anti-rabbit antibody (Bioworld, USA). Protein bands were detected and imaged with ChemiDocXRS+ gel imaging system (Bio-Rad, USA)and analyzed bydensitometric quantification using Image J software.
Gene set enrichment analysis with AdipoR2 expression
The gene expression profiles of glioma samples from CCGA were analyzed by GSEA15. Pearson's correlation was used to analyze the relationship between AdipoR2 and all identified genes with Matlabsoftware (P < 0.001). GSEA (http://www.broadinstitute.org/gsea/) analysis was used to identify pathway gene sets that are 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 is 50%. As a metric for ranking genes in the GSEA, the difference between the means of samples with low and high AdipoR2 expressionwas used, and the other parameters were set by their default values.
Kaplan-Meier survival analysis was used to estimatethe survival distributions. The log-rank test was usedto assess the statistical significance between stratifiedsurvival groups with use of GraphPad Prism. The t test was used to determine differences in each 2-group comparison.All statistical analyseswere performed using Matlab 2012, SPSS software for Windows, or GraphPad Prism (GraphPad Software). All data are presented asmean ± standard error. A 2-sided P value of < 0.05 was regarded as significant.