This study was approved by the ethics committee of the First Affiliated Hospital of Kunming Medical University.
Forty-four meningioma specimens were collected, including 38 cases of WHO grade I and 6 cases of WHO grade II, at the First Affiliated Hospital of Kunming Medical University. These specimens were collected from a total of 10 males and 34 females who had an average age of 49.75 ± 13.9 years. All samples were divided into three parts. One part was formalin fixed and paraffin embedded for IHC staining and Ki67 index detection. The second part was frozen at -80°C for PCR and Western blot assays. The third part was acutely dissected and isolated for primary meningioma cell culture.
Primary meningioma cell culture. Fresh meningioma tissues were minced into small pieces of approximately 1 mm3 and digested with 0.25% trypsin at 37°C for 18 minutes. The primary meningioma cells were collected and cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, BI, Israel) and 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA) . The primary cells were passaged when the cells reached 90% confluency. Early passage (less than five passages) primary cells were used for all designed experiments.
Immunohistochemistry (IHC). Fresh tissues were collected and fixed in 4% formaldehyde and embedded in paraffin. Tissue sections (5 µm) were deparaffinized, and antigens were retrieved by boiling the slides in 0.1 M citrate acid buffer for 5 min. Immunostaining for detecting TRβ1 (Clone J51, Santa Cruz Biotechnology, Inc., USA) was performed according to previously described protocols. Quantification of the immunostaining signal was performed based on the intensity and percentage of immunopositive cells.
Quantitative real‑time PCR. Total mRNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using Iscript™ cDNA reverse transcription reagent (BIO-RAD) to obtain cDNA for quantitative PCR analysis. The mRNA levels of the target genes were quantified using RT Real-Time™ SYBR Green PCR master mix (Thermo Fisher Scientific, USA) and detected with an ABI-7900 system (Applied Biosystems). The sequences of the primers were as follows: human TRβ1: F-5'-TACAGCCTGGGACAAACC-3', R-5'-GGCGACGACTGTTCATTT-3'; human GAPDH: F-5'-GGAGCGAGATCCCTCCAAAAT-3', R-5'-GGCTGTTGTCATACTTCTCAT GG-3'.
Cell proliferation assay. T3 was dissolved in 1 N NaOH solution and formulated into 1 ng/ml, 5 ng/ml, 10 ng/ml, and 20 ng/ml. The same concentration of NaOH was used as the negative control. Cells were seeded in a 96‑well plate at 4,000 cells/well in 100 µl of complete medium and cultured at 37°C. Twenty-four hours after plating, the culture medium was switched to complete medium supplemented with T3 or the NaOH control at the indicated concentrations (1 ng/ml, 5 ng/ml, 10 ng/ml, and 20 ng/ml). The MTT (5 mg/ml) reagent was added 48 hours after T3 intervention, and the OD value (490 nm wavelength) was measured with a spectrophotometric plate reader (Bio-Tek, USA). Cells were plated in 48-well plates at 0.2–12×105 cells/well. Twenty-four hours later, the cells were treated with either T3 (20 ng/ml) or the control at the indicated dosage for another 4 days. The cells were then fixed with 10% trichloroacetic acid (TCA) for 60 minutes at room temperature followed by washing with deionized water 5 times. The cells were stained with 0.4% (W/V) SRB for 15 minutes at RT followed by washing with 1% acetic acid 5 times. Finally, 10 mM Tris base was added to dissolve the dye, and the optical densities were measured at 530 nm with a spectrophotometric plate reader (Bio-Tek, USA). To determine the cell proliferation rate, cells were seeded on cell culture slides at 1×105 cells/well. Twenty-four hours after plating, the cells were treated with T3 (20 ng/ml) and EdU (1 ng/ml). Twenty-four hours later, EdU incorporation was measured using a Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen) following the manufacturer’s instructions. All experiments were performed in triplicate and repeated twice.
TRβ1 knockdown with siRNA. Meningioma cells were plated in six-well plates (1 × 106 cells/well) and transfected with siRNA targeting TRβ1 (GCGCTATGACCCAGA A AGT) or control siRNA (final concentration 20 nM; RiboBio, China) using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s protocol. Seven hours after transfection, the cell culture medium was replaced with fresh medium. The efficiency of siRNA-mediated gene silencing was detected 72 h after transfection by Western blotting analysis.
Western blot analysis. Frozen meningioma tissues or cells were collected and lysed in cold RIPA buffer supplemented with protease inhibitor cocktail (Sigma). Western blot analysis was performed as described previously . Briefly, 30 µg of each protein sample was subjected to SDS–PAGE and blotted onto polyvinylidene fluoride (PVDF) membranes. After incubation with specific primary antibodies at 4°C overnight, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 hour.
Statistical analysis. Data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 19.0 (SPSS, Inc. Chicago, IL, USA). Two‑tailed Student's t‑test was used for comparisons between two groups. P values < 0.05 were considered to be significant.