Human specimen collection
All tumor samples were obtained following transsphenoidal surgery performed at Beijing Tiantan Hospital. Fresh tumor samples were stored in liquid nitrogen. 63 tissue samples include 56 pituitary adenomas and seven normal pituitary glands. 56 pituitary adenomas were classified according to the 2017 WHO classification. In the present study, 56 cases include 10 cases of corticotroph adenomas (five silent ACTH and five functional ACTH), ten cases of gonadotroph adenomas, eight cases of somatotroph adenomas, eight cases of mammosomatotroph adenoma, five cases of lactotroph adenomas, ten cases of oncocytomas, and five cases of null cell adenomas.
This study was approved by the Ethics Committee of Beijing Tiantan Hospital (KY2013-015-02). Written informed consent was obtained from all of the enrolled subjects, and the study was performed in compliance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
For the gas chromatography–mass spectrum (GC–MS) analysis, tissue samples were mixed with 600 µl methanol/water (v/v 4:1) solution containing internal standards followed by thorough homogenization in a mixed grinding apparatus (MM400, Retsch, Germany) at 25 Hz for 2 min. Supernatants were lyophilized for subsequent oximation and silylation reactions. A QP 2010 GC–MS system (Shimadzu, Japan) with a DB-5 MS fused-silica capillary column (30 m × 0.25 mm × 0.25 µm, Agilent Technologies, Santa Clara, CA, USA) was used for the metabolic profiling. A pseudotargeted GC–MS metabolomics method was established elsewhere [11,12,13]. A total of 280 ion features assigned to 23 groups were defined for the data collection and quantification. The system parameter settings were previously described . Metabolite identities were determined based on commercial libraries (Mainlib, NIST, Wiley, and Fiehn) and an internal metabolite library.
Total RNA was extracted and purified using the mirVana™ miRNA Isolation Kit (Cat# AM1561, Ambion, Austin, TX, US) following the manufacturer’s instructions and checked for an RIN number to inspect the RNA integration using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US). Total RNA was amplified and labeled with the Low Input Quick Amp Labeling Kit, One-Color (Cat# 5190-2305, Agilent Technologies, Santa Clara, CA, US), following the manufacturer’s instructions. Labeled cRNA was purified using an RNeasy mini kit (Cat# 74106, Qiagen, GmBH, Germany). Each slide was hybridized with 1.65 µg Cy3-labeled cRNA using the Gene Expression Hybridization Kit (Cat# 5188-5242, Agilent Technologies, Santa Clara, CA, US) in a hybridization oven (Cat# G2545A, Agilent Technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 h of hybridization, the slides were washed in staining dishes (Cat# 121, Thermo Shandon, Waltham, MA, US) with the Gene Expression Wash Buffer Kit (Cat# 5188-5327, Agilent Technologies, Santa Clara, CA, US), following the manufacturer’s instructions. Slides were scanned by an Agilent Microarray Scanner (Cat# G2565CA, Agilent Technologies, Santa Clara, CA, US) with the default settings: dye channel = green, scan resolution = 5 µm, PMT 100%, 10%, 16 bit. Data were extracted with Feature Extraction software 10.7 (Agilent Technologies, Santa Clara, CA, US). The raw data were normalized using the quantile algorithm and limma packages in R.
Protein preparation and proteomic analysis
Proteins from pituitary adenoma tissues and normal pituitary glands were extracted using a total protein extraction kit (Millipore, Billerica, MA, USA). Protein concentrations were determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Proteins from each group of tissues were equally combined into a single pool as previously described . A mass of 100 μg of each pooled sample was then denatured, reduced and alkylated as described in the iTRAQ protocol (Thermo Fisher Scientific, Waltham, MA, USA) and digested overnight with 0.1 µg/µl trypsin solution at 37 °C. The digested pooled samples were subsequently labeled with 113, 115, 117, 118, 119 and 121 iTRAQ tags, respectively, according to the manufacturer’s protocol (AB Sciex, Framingham, MA, USA). The tagged peptides were dried via vacuum centrifugation and combined in one tube. Strong cation-exchange (SCX) chromatography was then performed as previously described [15, 16]. In brief, pooled samples were separated on an apoly-LC SCX column (4.6 × 250 mm, 5 μm, 100 Å) using an LC 100 instrument (Eksigent Technologies, Dublin, CA, USA), and then the labeled peptides were detected by ultraviolet radiation using SPD-20 (Shimadzu, Kyoto, Japan). A total of 48 fractions were collected, dried by speed vacuum centrifugation, and combined into 10 fractions according to the SCX chromatogram. Each fraction was injected onto a desalting column (350 µm × 0.5 mm, 3 µm C18, 120 Å) and separated on an analytical column (75 µm × 150 mm, 3 µm C18, 120 Å) using an Eksigent NanoLC instrument (Eksigent, Dublin, CA, USA). The samples were separated by capillary high-performance liquid chromatography and were subsequently analyzed using a Triple TOF 5600+ system (AB Sciex).
Protein identification and proteome annotation were performed using the ProteinPilot software package, version 4.5 (Applied Biosystems), and cross-referenced against the SwissProt database (March 2013) using the Mascot 2.2 search engine (Matrix Science, London, UK). The search parameters utilized to analyze the MS/MS data included: trypsin as the digestion enzyme with a maximum of two missed cleavages allowed; fixed modifications of carbamidomethyl (C) and iTRAQ Plex (K and N-terminus); variable modifications of oxidation (M); peptide mass tolerance of ± 20 ppm; fragment mass tolerance of ± 0.1 Da; and a peptide false discovery rate (FDR) of less than or equal to 0.01.
The GH3 rat pituitary cell line was purchased from the China Institute of Cell Line Resources and cultured in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, China) supplemented with 10% fetal bovine serum (Gibco, Auckland, USA) in a humidified incubator at 37 °C with 5% CO2. IDH2-targeted shRNA (TF702283A-D) was obtained from OriGene Clone (OriGene Technologies, MD, USA). Transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. A total of 1 µg of plasmid was used to transfect 1 × 106 GH3 cells.
Cell viability and Transwell assays
GH3 cells were transfected with shRNA or empty control vectors (noneffective 29-mer scrambled shRNA cassette in pGFP-C-shLenti vector). Cultures were adjusted to a density of 1 × 105 cells/ml, and 100 µl of cell suspension was plated into each well of a 96-well plate and cultured for 0 h, 24 h or 48 h before adding 20 µl of MTS tetrazolium solution to each well with incubation for an additional 4 h. Absorbance at 490 nm was measured using an ELISA plate reader (Thermo, USA).
Cell migration was assayed on fibronectin- and Matrigel-coated polycarbonate filters in modified Transwell chambers (Corning, USA). GH3 cells (5 × 104 cells) were introduced into the upper chambers. The time of incubation in the chambers was 24 h. Cells adhering to the lower membrane surface were fixed in 4% paraformaldehyde and stained with hematoxylin (Zhongshan Company, Beijing, China). The average number of migrated cells in five randomly chosen high-power fields was determined under light fields with fluorescent microscope (ZEISS, Jena, Germany). The assays were performed in triplicate.
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from GH3 cells with shRNA or empty control vectors using TRIzol reagent (Qiagen), and qRT-PCR was performed as previously described  with an Applied Biosystems 7500 Fast System (Life Technologies, Carlsbad, USA). The fold-change in differential expression for each gene was calculated using the comparative CT method (2−∆∆CT method) as previously described . GAPDH was used as the housekeeping gene. The primers are listed in Additional file 1: Table S1).
All data are presented as the mean ± standard error of the mean (SEM). One-way ANOVA was used for the comparisons of multiple groups. A p value less than 0.05 was considered statistically significant.