Cell culture
The immortalized normal lung cell line (BEAS-2B) and LUAD cell lines including NCI-H1975, H1299, A549 and HCC827 were cultured in RPMI-1640 or DMEM with 10% FBS and incubated at 37°C in a humidified incubator with 5% CO2. BEAS-2B was purchased from the Chinese Academy of Sciences Typical Culture Preservation Committee Cell Bank. A549 was purchased from Genetic Testing Biotechnology Corporation. HCC827 was gifted by Proteomics and Signal Transduction Center, Zhengzhou University. H1299 and NCI-H1975 were gifted by Henan Institute of Medical and Pharmaceutical Sciences, Zhengzhou University.
Reagents
Antibodies used included BCAT1 (Sangon Biotech, China), phospho-NF-κB p65 (Ser536) (CST, USA), IKKα/β (Wanleibio, China), IKBα (Wanleibio, China), p65 (Wanleibio, China), GAPDH (Sangon Biotech, China), β-Actin (proteintech, China), E-cadherin (proteintech, China), Vimentin (proteintech, China), Antibody cocktail to aerobic respiration (CST, USA). PDTC, L-Valine, L-Isoleucine, and L-Leucine (MCE, USA).
Immunohistochemistry (IHC) and Evaluation
The tissue microarrays (TMAs) consisting of 86 pairs of human LUAD and normal lung tissues were purchased from Outdo Biotech (China). This study was conducted with the informed consent obtained from all subjects. All experiments were performed in accordance with relevant guidelines and regulations of Declaration of Helsinki and has been approved by the Research Ethics Committee of Zhengzhou University. Anti-BCAT1 antibody was used for IHC detection of BCAT1 expression according to the manufacturer’s suggested protocols. In brief, paraffin sections were dewaxed by heat treatment in EDTA buffer, pH = 9. After blocking for 30 min with 3% bovine serum albumin (BSA), tissue sections were incubated with anti-BCAT1 antibody diluted 1:400 overnight at 4 °C. Then they were incubated with the secondary antibody (HRP labeled) at room temperature for 50 min. The staining intensity of tissues was scored as 0 (negative), 1 (1+), 2 (2+), 3 (3+). For statistical evaluation, tissues were scored as 0 (non-staining); 1, 1–25%; 2, 26–50%; 3, 51–75%; 4, 76–100% (positive staining). BCAT1 protein expression was evaluated as negative (0), weak positive (1, 2), positive (3, 4) according to the staining positive rate score. The total histological score was defined as the product of “staining intensity score” and “staining positive rate score”, and 0-5 indicated BCAT1 low expression while 6-12 indicated BCAT1 high expression.
Lentivirus transduction and Stable cell line construction
BCAT1 overexpression, shRNA and control lentiviral particles were purchased from Genechem and Hanbio Biotechnology (China). The shRNA sequences targeting BCAT1 were as follows: 5,-GGATCAAGAATGGGTCCCATATTCA-3,, The shRNA control sequence was: 5,-TTCTCCGAACGTGTCACGTAA-3,. A549 cells were seeded in 6-well plates and infected with BCAT1 overexpression, shRNA or control lentivirus respectively, followed by incubation overnight with 5 μg/mL polybrene (Sigma-Aldrich, USA). Stable BCAT1 knockdown and overexpression cells were selected with 2 μg/mL puromycin.
Protein extraction and Western blot
Cells were lysed in RIPA buffer (Solarbio, China) for 30 min at 4°C and then centrifuged at 12000 rpm for 15 min. The supernatant was collected and total protein was quantified using the BCA kit (Solarbio, China). Total protein was boiled and separated by 10% SDS–PAGE gel, and then transferred to PVDF membrane (Merck Millipore, USA). Membranes were blocked in 5% skim milk powder in TBS-T (TBS plus 0.5% Tween-20) at room temperature for 2 hours, and then blots were incubated with primary antibody at 4°C overnight. The membrane was probed with secondary antibody, and signals were detected with ECL Substrate (Beyotime Biotech, China) and Amersham Imager 600 System (General Electric Company, USA). Protein expression was quantified using Image J software, with GAPDH or β-Actin as an internal control.
RNA Extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cells with TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s instruction and cDNA was synthesized using the PrimeScript RT Reagent Kit (TaKaRa Clontech, China). qRT-PCR was performed using the ChamQTM Universal SYBR® qPCR Master Mix (Vazyme Biotech, China) and QuantStudio 5 Real-Time PCR System (Thermo Scientific, USA). The target mRNA expression level was quantified using the 2-ΔΔCt method with normalized to the endogenous gene ACTB. The following primer sequences were used for qRT-PCR: BCAT1 forward, 5,-TGGAGAATGGTCCTAAGCTG-3,, reverse, 5,-GCACAATTGTCCAGTCGCT C-3,; NFKBIB forward, 5,-GCCCTGACCGTACTCCCGAC-3,, reverse, 5,-GGCCTCC ACTGCCAAATGAA-3,; BCKDK forward, 5,-CTTCTGAAAAGTGCTCGGTAC-3,, reverse, 5,-CTTCTGGAAGGCACGGATATAT-3,; BCKDHA forward, 5,-GAGGCAGG TGTGCTGATGTATCG-3,, reverse, 5,-AGTGGAGAGGAGATAGTGACGAAGTG-3,; BCKDHB forward, 5,-GGTGGCTCATTTTACTTTCCAG-3,, reverse, 5,-AGGATCTTT GGCCAATGAGTTA-3,; ECHS1 forward, 5,-GTGATATCATCTATGCCGGTGA-3,, reverse, 5,-GTCTCAACAGGACAAATCTTGC-3,; GAPDH forward, 5,-CTCAAGGG CATCCTGGGCTA-3,, reverse, 5,-CGTCAAAGGTGGAGGAGTGG-3,; ACTB forward, 5,-GGCATCCACGAAACTACCTT-3,, reverse, 5,-CTCCTGCTTGCTGATCCACA -3,. Primers used for qRT-PCR were synthesized by Shenggong, China.
RNA sequencing (RNA-seq) and Informatics analysis
RNA-seq was performed with BCAT1 overexpression and control A549 cells (each with three replicates) using BGISEQ-500 sequencing system. In brief, RNA sample was extracted and the quality was assessed, followed by generation of cDNA libraries. Gene expression levels for each sample were quantified using the FPKM method (fragments per kilobase of transcript per million mapped reads) and processed for analysis. DEGs were screened by fold change ≥ 1.5 and the adjusted p value ≤ 0.01. Gene Ontology (GO) annotation proteome was performed from the UniProt-GOA database (http://www.ebi.ac.uk/GOA). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was based on database (http://www.genome.jp/kegg/).
Cell proliferation assay
The cell proliferation ability was measured by using the xCELLigence Real-Time Cell Analyzer (RTCA)-MP system (Acea Biosciences, USA), which provided a continuous and quantitative measurement of the cell index to monitor cellular growth status. First, 50 μL of culture medium was added in each well of E-Plate 12 (Roche Applied Science, USA) to detect the baseline. Then cells were suspended in 100 μL medium and seeded in E-Plate 12 with a density of 5×104 cells/mL. After 30 min, the E-Plate 12 was inserted into RTCA-MP device and incubated at 37°C with 5% CO2. Cell index was measured every 5 min to reflect the cellular proliferation ability.
CCK-8 assay
Cells in the logarithmic growth phase were seeded in a 96-well plate (3000 cells/well), and the total volume of cell suspension per well was 100 μL. After incubation for 24 h and 48 h, 10 μL CCK-8 reagent was added into each well, and then cells were incubated for 1-4 h at 37°C in the dark. The absorbance (A) value at 450 nm wavelength was detected with a microplate reader (Thermo, USA).
Colony formation assay
Cells were seeded into 6-well plates (1000 cells/well). Then cells were cultured at 37°C with 5% CO2 for approximately one week. After that, clones were washed with PBS and then fixed with 4% paraformaldehyde, followed by staining with 1% crystal violet for 30 min. The colonies were examined and counted with a microscope.
Wound-healing assay
Cells were seeded with a density of 106 cells/well in 6-well plates, when the cell density reached 95%, monolayers were scratched with a 10 µL pipette tip to create scratch wounds. The suspended cells were washed away with PBS, and then cultured in RPMI-1640 medium with 2% FBS. The distance between scratches was measured at 0 h, 12 h, 24 h, and 48 h, and the relative residual area (%) was calculated using the following equation: (current gap distance/original gap distance) x 100%.
Cell migration and invasion assays
For migration assay, 5×104 cells were suspended in serum-free medium and seeded in the top chambers, then the lower chambers were filled with 600 µL medium containing 10% FBS. After incubation for 8~12 h, the migratory cells were fixed with methanol and stained with a 0.1% crystal violet solution. Then cells were counted with a microscope (magnification, x200; Olympus BX53, Japan). For invasion assay, the difference was that Matrigel (BD Biosciences, USA) was diluted in serum-free medium (1:6) and added to the upper chamber before cells were seeded.
Xenograft mice model
Animal study was performed in accordance with the guidelines of the National Act on the Use of Laboratory Animals (P. R. China), approved by the Animal Ethics Committee of Zhengzhou University. This study was reported in accordance with ARRIVE guidelines. BALB/c nude mice (female, aged 4 weeks) were upraised in SPF conditions in Henan Key Laboratory for Pharmacology of liver diseases, with a 12 h light/dark cycle and free access to food and water. BCAT1 overexpression and control A549 cells (5x106 in 100 μL PBS) were subcutaneously injected into the flanks of mice, respectively. Five weeks after injection, mice were sacrificed by cervical dislocation and the tumors were collected for analysis. Tumor volume was evaluated with the formula: tumor volume = (width2 x length)/2.
Cellular oxygen consumption rate (OCR) detection
Briefly, cells (8 x 104) were seeded into a 96-well plate and then cultured at 37°C with 5% CO2 for overnight. Next, the BBoxiProbeTM R01 (BestBio, China) and oxygen blocking fluid were added into the 96-well plate chambers in sequence at 37℃, and the fluorescence intensity at the excitation wavelength of 468 nm was detected by the CLARIOstar Plus (BMG, Germany).
Statistical analysis
All statistical analyses were performed using SPSS 21.0 and visualized with GraphPad Prism 6.0. Data are shown as mean ± standard error of measurement (SEM). A p value ≤ 0.05 was considered as statistically significant.