We retrospectively reviewed pituitary adenoma patients in Beijing Tiantan Hospital from January 1, 2005 to December 31, 2017. All patients were classified according to preoperative images, including hormone, plain and enhanced head MRI, thin layer skull base CT scanning and three-dimensional reconstruction. Patients who suffered from meningioma and pituitary adenoma simultaneously or successively were included in this study. The present study was conducted in accordance with established ethical guidelines as outlined in the Declaration of Helsinki. We obtained written informed consent from all participants, and the Ethics Committee of Beijing Tiantan Hospital approved this study.
ceRNA microarrays and construction of pathway act network
ceRNA means competing endogenous RNA. ceRNA microarray includes mRNA, lncRNA and circRNA. 5 pituitary adenomas of PAM and 5 SPAs were performed ceRNA microarray (Additional file 1: Table S1). Total RNA was extracted and purified using a mirVana™ miRNA Isolation Kit without phenol (Cat # AM1561, Ambion, Austin, TX, US). RNA samples from each group were then used to generate fluorescence-labeled cRNA targets for the SBC human ceRNA array V1.0 (4 × 180 K). The labeled cRNA targets were then hybridized with the slides. After hybridization, the slides were scanned on an Agilent Microarray Scanner (Agilent Technologies, Santa Clara, CA, US). The data were extracted with Feature Extraction software 10.7 (Agilent Technologies, Santa Clara, CA, US). Raw data were normalized by the Quantile algorithm using the limma package of the R program. The microarray experiments were performed according to the protocol of Agilent Technologies, Inc. at Shanghai Biotechnology Corporation. Ratios were calculated between the PAM and SPA. Then, differentially expressed genes were identified by using the t-test with a cut-off criteria of P < 0.05 and fold-change > 2 or < 0.5.
The selected mRNAs were grouped in functional categories based on the Gene Ontology database (GO: http://www.geneontology.org/). We identified the significant pathways of the differentially expressed genes by using IPA software (http://www.ingenuity.com). We used the software Cytoscape software (V2.8.0) (http://www.cytoscape.org) to construct a pathway act network for graphical representations of central pathways using the genes enriched in the significant canonical pathways of IPA (P < 0.05).
Quantitative real-time PCR validation
Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and then reversed transcribed using a HiFiScript gDNA Removal cDNA Synthesis Kit (CWBio, China) according to the manufacturer’s instructions. Subsequently, we performed qRT-PCR using SYBR Green assays in a total reaction volume of 10 μl which was performed on an ABI 7500 System (Applied Biosystems). GAPDH was used as a reference gene. For the quantitative analysis, expression levels were calculated based on CT values (corrected for GAPDH expression) according to the equation: 2−∆CT [∆CT = CT (gene of interest) − CT (GAPDH)]. All qRT-PCR analyses were performed in triplicate. Student’s t-tests were applied, and a P-value < 0.05 was considered significant. The primer sequences are presented in Additional file 2: Table S2.
Short interfering RNA transfection of HEK 293T cells
293T cells were authenticated by China Infrastructure of Cell Line Resource and tested negative for mycoplasma according to China Infrastructure of Cell Line Resource. Short interfering RNA (siRNA) against MEN1 (si-MEN1) and the negative control (sh-NC) were synthesized by RiboBio (Guangzhou, China) and employed (SiMEN1 CTACGACGGCATCTGCAAA). Exponentially growing HEK 293T cells (2 × 105) were seeded onto 6-well plates overnight, and then transfected using Lipofectamine®3000 transfection reagent (Thermo Fisher Scientific, Massachusetts, USA) (final concentration: siRNA or negative control: 50 nM). The transfection efficiency was determined by qRT-PCR at 48 h after transfection. The protein extracted from transfected cells was collected to measure the level of MEN1and mTOR pathway genes by Western blot at 72 h after transfection.
Transfected 293T cells were lysed in nondenaturing lysis buffer (Applygen). For Western blotting, the protein samples (30 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. Different blots were incubated with antibodies against MEN1 (ab 92443, 1:5000, Abcam, USA), phosphor-AKT(1:1000, AF0016, Affinity, China), AKT(1:500, 10176-2-AP, Proteintech, USA), phospho-mTOR(1:500, AF3308, Affinity, China), mTOR(1:300, 20657-1-AP, Proteintech, USA) or vinculin(1:10,000, Abcam, USA), followed by incubation with secondary antibodies tagged with horseradish peroxidase (Santa Cruz Biotechnology). The blots were visualized by enhanced chemiluminescence, and densitometry was performed with an imaging apparatus (Amersham Imager 600, GE). Vinculin was used as a loading control.
Isolation and culture of primary pituitary adenoma and meningioma cells of PAM
Before isolating primary pituitary adenoma and meningioma cells, all pituitary adenoma and meningioma biopsies were washed in PBS supplemented with P/S and 10% FBS in order to remove unhealthy tissues. After carefully washing, the remaining healthy tissues were cut into small pieces using refined scissors and dissociated into small cell clumps (10–50 cells/clump) with pre-chilled accutase. Then these small clumps of primary pituitary adenoma and meningioma cells were collected and re-suspended in cell culture medium: DMEM/F12 (Invitrogen), 1% Knock Out serum replacement (KSR, Invitrogen), N2 supplement (100X, Invitrogen), B27 supplement (100X, Invitrogen),1 mM l-Glutamine, 0.1 mM NEAA, 0.1 mM 2-ME with 4 ng/ml bFGF (R&D systems), and 10 ng/ml EGF (R&D systems). After evaluating the cell number and viability, primary pituitary adenoma and meningioma cells were seeded at 2 × 105 cells/well (6 well plate) on Matrigel-embedded plate. The medium was replenished every 24 h for 5–7 days before passaging. Pituitary adenoma and meningioma cells can be passaged by accutase digestion and can be routinely maintained in cell culture medium for 5–10 passages.
Apoptosis induction assays
For the human FAS;CD95 ELISA assay, pituitary adenoma cells were plated into 96 well plate at 2 × 104 cells/well and treated with rapamycin at 0 nM, 0.5 nM, 1 nM, 2 nM. and Meningioma cells were plated into 96 well plate at 2 × 104 cells/well and treated with rapamycin at 0 nM, 1 nM, 2 nM, 5 nM, 10 nM. Supernatants were collected at 48 h and 72 h for apoptosis detection. Apoptosis index was measured following the instruction of the human FAS;CD95 ELISA kit (KS4622, Keshun biotechnology).
For the annexin V Conjugates based assay, pituitary adenoma cells were plated into 96 well plate at 2 × 106 cells/well and treated with rapamycin at 0.5 nM, 1 nM, 2 nM. Meningioma cells were plated into 96 well plate at 2 × 106 cells/well and treated with rapamycin at 2 nM, 5 nM, 10 nM. Supernatants were collected at 48 h and 72 h for apoptosis detection. Cells were then treated with Annexin V Alexa Fluor 488 to identify apoptotic cells by flow cytometry.
Statistical analysis was performed using SPSS software (version 20.0, IBM). The χ2 test was used to analyze metric variables. The two independent-samples t test was used to test ordinal variables. P-values of < 0.05 were defined as a significant difference.