Cell culture and reagents. NMC cell lines, Ty82 (RIKEN Cell Bank), HCC2429 cells (UT Southwestern Medical Center) and A549 lung adenocarcinoma cells (American Type Culture Collection) were incubated in RPMI-1640 medium supplemented with 10% FBS and antibiotics at 37˚C with 5% CO2 [17, 18]. BET inhibitors, JQ-1 and OTX015 (MK8628; Birabresib) and the WEE1 G2 checkpoint kinase (WEE1) inhibitor, AZD1775 (MK1775; Adavosertib) were purchased from Selleck Chemicals. The BET-inhibitor-resistant HCC2429 cell line was established by exposure to increasing concentrations of JQ-1 (1-100 nM) over a 3-months period, as described previously [19].
MTS assay. Cell proliferation assays were performed using the CellTiter 96 Aqueous One Solution assay (Promega Corporation). Briefly, cells were seeded into 96-well plates (3-5x103 cells/well) overnight in triplicates. Following the incubation, the cells were treated with the indicated concentrations of drugs for 3 days, then the assays were performed. The experiments were performed independently at least three times.
Transfection. Stealth RNAi small interfering (si)RNA (Thermo Fischer Scientific, Inc.) was used for NUTM1 knockdown, Specifically, NUTM1-siRNAs (cat. no. HSS138007, HSS138008 and HSS138009) and Stealth RNAi siRNA Negative Control (cat. no. 12935300, Thermo Fischer Scientific, Inc.) were used. Lipofectamine® RNAiMAX transfection reagent and Opti-MEM I reduced serum medium (Thermo Fischer Scientific, Inc.) were used for siRNA transfection. The cells were treated with 50 pmol of siRNA for the indicated time periods, and subsequent experiments were then performed.
miRNA function analysis was performed using miRCURY LNA miRNA mimics and inhibitors (all from Qiagen, Inc.); specifically hsa-miR-21-5p inhibitor (cat. no. YI04100689), hsa-miR-21-5p mimic (cat. no. YM00473093), miRNA inhibitor controls, and miRNA mimic controls. Lipofectamine® 3000 (Thermo Fischer Scientific, Inc.) and Opti-MEM I reduced serum medium were used for miRNA transfection. Cells were plated into 96-well plates and treated with 0.66 pmol of miRNA mimics or 5 pmol of miRNA inhibitors for 72 h, and subsequent assays were performed.
Western blotting. NUT (cat. no. 3625), β-actin (cat. no. 4967), and horseradish peroxidase (HRP)-labeled anti-rabbit IgG (cat. no. 7074) antibodies were purchased from Cell Signaling Technology, Inc. Trimethylation of lysine 36 on histone H3 (H3K36me3; cat. no. ab9050) and Histone H3 (cat. no. ab1791) antibodies were purchased from Abcam. Proteins were extracted from the cells using cell lysis buffer (Csll Signalint Technology, Inc.) supplemented with complete protease inhibitor cocktail (Roche Diagnostics). All antibodies were used at a dilution of 1:1,000. NuPAGE gels, Pierce Power Blotter, and iBind Western system (Thermo Fischer Scientific, Inc.) were used for western blotting, according to the manufacturers’ protocols. Protein expression was measured using the WSE-6100 LuminoGraph I (ATTO Corporation) and quantified using the CS Analyzer version 4 (ATTO Corporation).
Next-generation sequencing. Genomic DNA was extracted from the cell lines using the Blood & Cell Culture DNA mini kit (Qiagen, Inc.) and from patient-derived tumors using the QIAamp DNA FFPE Tissue kit (Qiagen, Inc.). DNA was quantified using the Qubit 2.0 fluorometer with the Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Inc.). A total of 50 ng of DNA from cell lines and 10 ng of DNA from tissue samples were used for PCR amplification using the Ion AmpliSeq Library kit 2.0, and Ion AmpliSeq Comprehensive Cancer Panel (Thermo Fischer Scientific, Inc.). The Ion Express Barcode Adapters (Thermo Fischer Scientific, Inc.) were ligated to the PCR products for barcoding the tissue samples. AMPure XP beads (Beckman Coulter, Inc.) were used for PCR product purification. The libraries were sequenced using an Ion PGM system (Thermo Fischer Scientific, Inc.). DNA sequencing data were obtained using the Torrent Suite version 4.0 software (Thermo Fischer Scientific, Inc.). Variant caller version 4.0. was used to call variants. The reads were aligned with the GRCh38 reference genome, and Ion Reporter version 5.0 and CLC Genomics Workbench version 9.5.1 (Qiagen, Inc.) were used for additional analysis.
Small RNAs were extracted from the NMC cell lines using a PureLink miRNA isolation kit (Thermo Fischer Scientific, Inc.) and quantified using the Agilent 2100 Bioanalyzer with an Agilent RNA small RNA kit (Agilent Technologies, Inc.). A small RNA library was prepared using the Ion Total RNA-Seq kit v2 (Thermo Fischer Scientific, Inc.), and the libraries were sequenced on the Ion PGM system. The reads were aligned with miRBase miRNAs. miRNA expression was analyzed using the CLC Genomics Workbench version 9.5.1.
miRNA assay. Small RNAs were extracted from NMC cells using the PureLink miRNA Isolation kit and quantified using a NanoDrop spectrophotometer (Thermo Fischer Scientific, Inc.). miRNA expression was analyzed using TaqMan microRNA assays (Thermo Fischer Scientific, Inc.), mir-21-5p (cat. no. 000397) and RNU48 (cat. no. 001006). In the present study, RNU48 was used as a housekeeping miRNA. Reverse transcription (RT) was performed using the TaqMan microRNA Reverse Transcription kit (Thermo Fischer Scientific, Inc.) and the indicated RT primers in the assay kits. PCR was performed using the QuantStudio 3D Digital PCR Master Mix, QuantStudio 3D Digital PCR 20 K Chip kit v2, and ProFlex 2x Flat PCR system (Thermo Fisher Scientific, Inc.). PCR was performed according to the manufacturer’s instructions [20]. Absolute quantification was performed using the QuantStudio 3D Digital PCR system (Thermo Fischer Scientific, Inc.). We analyzed the data using the QuantStudio 3D AnalysisSuite Cloud Software (Thermo Fischer Scientific, Inc.).
Detection of the BRD4-NUT fusion genes in clinical samples. The Medical Ethics Committee of the Asahikawa Medical University approved the clinical sample analysis (approval no. 15218-2). All methods were performed in accordance with the relevant guidelines and regulations. Clinical samples [formalin-fixed paraffin-embedded (FFPE) tissues] from patients with a thoracic tumor were collected at the Asahikawa Medical University Hospital. Written informed consent was obtained from all patients before the tumor sample collection. The specific primers and probes for BRD4-NUT detection were designed and the sequences were: BRD4-NUT forward, 5'-TGAAGGGCTTCTCGTCCTCAG-3' and reverse, 5'-GCGGCACTAGGTTTCATGCTC-3'; and BRD4-NUT FAM probe, 5'-TCGGAGAGCTCAGTGAGTCCAGCT-3'. An RNeasy Mini kit (Qiagen, Inc.) was used to extract RNA from the cell lines and the RecoverAll Total Nucleic Acid Isolation kit for FFPE (Thermo Fischer Scientific, Inc.) was used to extract RNA from the clinical samples. First-strand cDNA was synthesized using the SuperScript VILO cDNA Synthesis kit (Thermo Fischer Scientific, Inc.), and the RT reaction was performed according to the manufacturer’s protocol. The QuantStudio 3D Digital PCR Master Mix, QuantStudio 3D Digital PCR 20 K Chip Kit v2, and ProFlex 2x Flat PCR System were used for PCR amplification according to the manufacturer’s protocol. Absolute quantification was performed using the QuantStudio 3D Digital PCR system, and QuantStudio 3D Analysis Suite Cloud software was used for data analysis [21].
Statistical analysis. Data are presented as the mean ± SEM. Statistical analysis was performed using the GraphPad Prism Software version 7.0 (GraphPad Software, Inc.), and the results were analyzed using a Student’s t-test, One-way ANOVA, and Tukey’s multiple comparisons test. A Two-sided P < 0.05 was considered to indicate a statistically significant difference.