2.1 Cell culture and treatment

Human thyroid follicular epithelial cell line Nthy-ori-3-1 provided by European Collection of Cell Cultures (ECACC) and thyroid cancer cell lines (TPC-1, BCPAP and KTC-1) purchased from BeNa Culture Collection (BNCC) were all incubated in Roswell Park Memorial Institute (RPMI)-1640 medium (VivaCell, Shanghai, China) with 10% fetal bovine serum (FBS; VivaCell, Shanghai, China) with 5% CO2 at 37 °C in a humidified incubator. The cells were passaged every three days, and log-phase cells were collected for experiments.

Additionally, all cells were treated by midazolam (Nhwa Pharmaceutical, Jiangsu, China) at varying doses (5, 10, 15, 20 and 40 μg/ml) for 24 h [14]. Subsequently, PTC-1 cells were treated by midazolam (15, 20 and 40 μg/ml) for 24 h and KTC-1 cells were treated by midazolam (35, 50 and 75 μg/ml) for 24 h.

2.2 Gene interference

The siRNAs targeting YWHAH (siRNA-YWHAH-1/2) and the scramble siRNA (siRNA-NC) were designed by Integrated DNA Technologies (San Diego, CA, USA). The synthetic YWHAH overexpression plasmid (Ov-YWHAH) and Ov-NC were purchased from Zoman (Beijing, China). Cells were seeded in 24-well plates at an optimized concentration of ~ 1 × 105 cells/well. When cell confluence had reached 60–70%, the aforementioned plasmids and siRNAs (100 nM) were then transfected into cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) at 37 °C for 48 h according to the manufacturer’s instructions. 48 h post transfection, the cells were collected for further assays. The following siRNA sequences were used: siRNA-YWHAH-1, 5′-CTCCAATGAAGATCGAAATCTCC-3′; siRNA-YWHAH-2, 5′-CACTAAACGAGGATTCCTATAAG-3′.

2.3 Cell counting kit-8 (CCK-8)

Nthy-ori-3-1 cells and thyroid cancer cells were seeded into the 96-well flat-bottomed plate (5 × 103 cells/well) and cultured at 37˚C, and treated by midazolam (5, 10, 15, 20 and 40 μg/ml) for 24 h. Additionally, TPC-1 cells were transfected with siRNA-YWHAH, siRNA-NC or Ov-YWHAH, Ov-NC in the presence or absence of midazolam (40 μg/ml). 10 μl CCK8 working liquid (ApexBio Technology, Houston, TX, United States) was added to each well. The plates were incubated for 2 h and the absorbance was measured with a microplate reader (Thermo MK3, Thermo Fisher Scientific, USA) at 450 nm.

2.4 5-Ethynyl-2′-deoxyuridine (EDU) staining

As per the protocol of the EdU Labeling Kit (Beyotime, Shanghai, China), TPC-1 and KTC-1 cells were seeded into 12-well plates (1.5 × 104 cells/well) were incubated with pre-warmed complete medium consisting of 10 µM EDU working solution for 1 h. Afterwards, cells were fixed with 3.7% paraformaldehyde for 15 min were then permeabilized by 0.5% Triton X-100 for 20 min. Following cultivation with 1 × ClickiT® reaction cocktail, the cell nuclei were stained with DAPI. The images were captured under a fluorescence microscope and the EDU-positive cells were counted with ImageJ software (version 1.8.0; National Institutes of Health).

2.5 Wound healing assay

TPC-1 and KTC-1 cells were plated in 6-well plates at 5 × 105 cells/well using serum-free RPMI-1640 medium at 37˚C for 24 h until cells reached 90% confluence. Linear wounds were created using a pipette tip, followed by PBS washing to remove the detached cells. Subsequently, the obtained cells were cultured in RPMI-1640 medium containing 2% FBS for 24 h at 37 °C. The wound healing rates were observed under an optical microscope at 0 and 24 h under a light microscope. The migration rate was calculated based on the formula: (Wound width at 0 h-wound width at 24 h)/wound width at 0 h × 100%.

2.6 Transwell assay

Transwell invasion assays were performed using 8-mm pore size Transwell® plates (Corning Inc.) that were coated with Matrigel (BD Biosciences) for 1 h at 37 °C. TPC-1 and KTC-1 cells (5 × 104 cells) were seeded into the upper chamber of the Transwell plate. The upper wells were filled with serum-free RPMI 1640 and the bottom wells were filled with RPMI-1640 containing 10% FBS. The cells were fixed with 10% methanol at room temperature for 10 min and stained with 0.5% crystal violet at room temperature for 10 min. The invaded cells were counted under a light microscope.

2.7 Flow cytometry assay

Cell apoptosis was appraised utilizing Annexin V-FITC/PI apoptosis detection kit (Qihai Biotec, Shanghai, China). TPC-1 and KTC-1 cells were seeded at a density of 2 × 105 cells per well in six-well plates. After being digested with trypsin, cells were centrifuged at 1000Xg for 5 min and re-suspended in 100 µl 1X binding buffer. Then the cells were double-stained with 5 μl Annexin V-FITC and propidium iodide (PI) for 15 min in the dark. The analysis was performed using a BD FACSAria™ II flow cytometer (Becton–Dickinson and Company), and the data were analyzed using CellQuest Pro software (version 5.1; Becton–Dickinson and Company).

2.8 Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was isolated from TPC-1 and KTC-1 cells using TRIzol reagent (TSINGKE, Shanghai, China). mRNA was reverse transcribed into cDNA using the cDNA synthesis Kit (TSINGKE, Shanghai, China) and the reaction was incubated at 25 °C for 5 min, 42 °C for 30 min, 85 °C for 5 min and then kept at 4 °C for 5 min. PCR reaction was implemented using 2 × TSINGKE® Master qPCR Mix (TSINGKE, Shanghai, China). The thermocycling conditions were as follows: Initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation (45 s at 95 °C), annealing (45 s at 60 °C) and extension (8 min at 68 °C), before a final extension at 68 °C for 10 min. GAPDH was used for normalization, where gene expression was calculated using the 2−ΔΔCq method. The following primers were used: YWHAH forward, 5′-CCGCTACTTAGCAGAGGTCG-3′ and reverse, 5′-TGGCATCATCGAAGGCTTGT-3′ and β-actin forward, 5′-TGGAGTCCACTGGCGTCTTC-3′ and reverse, 5′-GCTTGACAAAGTGGTCGTTGAG-3′.

2.9 Western blot analysis

Total protein was extracted from TPC-1 and KTC-1 cells containing 1% protease and 1% phosphatase inhibitor cocktail (Sigma Aldrich; Merck KGaA) on ice using RIPA buffer (Abiowell, China). The concentration of protein samples was measured using a BCA kit (GENEray, Shanghai, China). Subsequently, equal amounts of proteins (40 µg) were separated by 10% SDS-PAGE and then transferred onto PVDF membranes. The membranes were blocked by 5% skim milk at 4 °C at room temperature for 1 h, and then incubated with primary antibodies against MMP2 (ab92536; 1/1000; Abcam), MMP9 (ab76003; 1/1000; Abcam), BCL2 (ab182858; 1/2000; Abcam), Bax (ab32503; 1/1000; Abcam), cleaved caspase3 (ab32042; 1/500; Abcam), YWHAH (ab206292; 1/1000; Abcam) and GAPDH (ab9485; 1/2500; Abcam) overnight at 4 °C at room temperature. The membranes were washed three times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (ab6721; 1/2000; Abcam) for 1 h at room temperature. The protein blots were visualized with the ECL solvent (Qihai Biotec, Shanghai, China) and quantified by ImageJ software (version 1.8.0; National Institutes of Health).

2.10 Immunofluorescence staining

TPC-1 and KTC-1 cells (2 × 105 cells/ml) were fixed with 4% paraformaldehyde at room temperature for 15 min and permeabilized with 0.5% Triton X-100 for 20 min at room temperature. After being sealed by 5% BSA, cells were incubated with primary antibodies against cleaved caspase3 overnight at 4 °C and secondary antibody conjugated with Alexa-Fluor 488 for 1 h at room temperature. The cell nuclei were stained with 1 mg/ml DAPI and the images were captured under a fluorescence microscope.

2.11 Statistics

All experiments were performed at least three times. All data were presented as mean ± standard deviation (SD). Statistical analyzes were performed using GraphPad 6 Software (GraphPad Software, Inc.). Multiple group comparisons were analyzed with one-way ANOVA along with Tukey’s post hoc test. P-value less than 0.05 was considered to indicate a statistically significant difference.

2.12 Bioinformatics tools

The midazolam structure and YWHAH protein crystal structure (PDB ID: 2c63) were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and RCSB (http://www.rcsb.org/) databases, respectively. CB-Dock2 server (https://cadd.labshare.cn/cb-dock2), which is a molecular docking program based on AutoDock Vina [15], was applied to explore the docking of midazolam with YWHAH. All parameters were set to their default values.