Cell lines and cell culture
H1299, HLF (HLF-a) and HBE cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). A549 and H460 cells were cultured in RPMI1640 medium supplemented with 10% FBS. The cells were grown at 37 °C in an atmosphere of 5% CO2. All the cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA).
The NMI overexpression plasmid pEZ-Lv203-NMI (Catalog No.: EX-K2261-Lv203) and its control vector pEX-NEG-Lv203 (Catalog No.: EX-NEG-Lv203), NMI shRNA plasmid psi-LVRH1GP-NMI (Catalog No.: HSH022093-LVRH1GP) and its control vector psi-LVRH1GP (Catalog No.: CSHCTR001–1-LVRH1GP) were obtained from GeneCopoeia (Rockville, MD). The target sequence of NMI shRNA1 is 5′-gagtgcagtcatcacgttt-3′, and the target sequence of NMI shRNA2 is 5′-ctaggtcaacctcacatag-3′. A549 and H1299 cells were transfected with corresponding plasmids to overexpress or knock down NMI.
The cells (2 × 105/ml) seeded in 6-well plates overnight were mixed gently 4 μg shRNA or plasmids and 10 μl of Lipofectamine 3000 (Invitrogen, Carlsbad, CA) in 250 μl opti-MEM (Gibco, Gaithersburg, MD) and incubated at 37 °C for 48 h.
Lysate preparation from tumor tissues
Lung cancerous tumors and adjacent tissues were obtained from 20 lung adenocarcinoma patients who underwent surgery therapy at the First Affiliated Hospital of Dalian Medical University between 2014 and 2015. The surgery and the study had been approved by the medical ethics committee at the First Affiliated Hospital of Dalian Medical University, and the informed consents were obtained from patients in accordance with the Declaration of Helsinki and with institutional guidelines. The tissues (100 mg) were washed with PBS to remove blood, transferred to liquid nitrogen immediately and homogenized thoroughly with RIPA buffer with protease inhibitor. After incubation on ice for 30 min, these tissues were sonicated for 2 to 5 min at power of about 180 W. The lysates were centrifuged at 12,000 g for 20 min at 4 °C, and the supernatants were collected.
Protein lysates (40 μg) were separated by 4% to 12% SDS-PAGE, electrophoretically transferred to PVDF membranes, immunoblotted at 4 °C overnight with antibodies against NMI, p300 (Santa Cruz Biotechnology, Santa Cruz, CA), β-actin, COX-2, p-PI3K, PI3K p85 (Tyr458)/p55 (Tyr199), Akt, p-Akt (Ser473), p-PDK1, p-GSK3β, p110 β, P38, p-P38, ERK1/2, pPARP, pTyr202/Y204-ERK1/2, cleaved caspase-3, cleaved caspase-9, MMP2, MMP9, E-cadherin, B-cadherin (all purchased from Cell Signaling Technology, Beverly, MA), Bcl2 (Proteintech, Wuhan, China), followed by incubation with HRP-conjugated second antibody, and finally detected by enhanced chemiluminescence.
Three days after transfection, cell culture media were collected. The amounts of PGE2 in the media were determined by Human Prostaglandin E2 (PGE2) ELISA Kit (Bluegene Biotech).
Analysis of promoter activity
The promoter of human COX-2 gene (−891 to +9) was truncated to 6 different lengths. Each fragment was inserted into a luciferase reporter vector pGL3. Luciferase reporter assays were performed using the kit Dual-Luciferase® Reporter Assay System (Promega, Madison, WI).
Cell viability assay
Cells were seeded into 96 well plates (4 × 103 cells per well). Cell viability was measured by MTT assay 48 h after transfection.
Cells were transfected with NMI overexpression or shRNA plasmid. After 48 h, cells were trypsinized, harvested, washed with PBS twice, and resuspended in Annexin V Binding Buffer at a concentration of 0.25–1.0 × 107 cells/ml. 100 μl cell suspension was transferred to a 5 ml test tube, and 5 μl of APC Annexin V was added in, followed by the addition of 5 μl 7-AAD Viability Staining solution, per instructions of the Annexin V APC/7AAD staining kit (Keygen biotech, Jiangsu, China). Cells were pipetted up and down and incubated for 15 min at room temperature in the dark. Finally, 400 μl Annexin V Binding Buffer was added to each tube before the cells were analyze by flow cytometry (Beckman Coulter, Brea, CA). Apoptosis was measured in terms of the 7AAD-positive cells.
Cells were seeded in 6-well plates (4 × 105 cells per well), and were grouped as (a) PBS control, (b) LacZ control, (c) NMI overexpression (d) control shRNA, (e) NMI shRNA1, (f) NMI shRNA2. 12 h after transfection, cells were scratched with sterile 200 μl tips, and photographed at 0 h, 48 h and 72 h.
Cells were incubated on chamber slides in 6-well plates with or without NMI shRNA transfection. The samples were fixed with 4% polyoxymethylene for 30 min at room temperature, permeabilized with PBST (PBS with 0.2%Triton X-100), blocked with bovine serum albumin (BSA) for 30 min and incubated with NMI and p300 antibodies (1:200 dilution) overnight at 4 °C. After washed 3 times, cells were incubated with the fluorescein isothiocyanate and rhodamine conjugated secondary antibodies for 1 h. The nuclei were counterstained with 40,6-diamidino-2-phenylindole (DAPI). The images were taken by Olympus confocal laser scanning microscope.
Nuclear protein (1 mg) was used for each IP. The proteins were pulled down with Protein A/G sepharose and then detected by Western blot analysis with specific antibodies.
Female nude mice (4 to 5 weeks old) were maintained in SPF laboratory Animal Central. All animals’ maintenance and procedures were carried in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, and passed through the training progress and approval by Animal Care and Ethics Committee of Sun Yat-sen University Cancer Center. Each nude mouse was injected with 2 × 105 human A549 cells suspended in 100 μl PBS, subcutaneously near the axillary fossa. The mice were randomly divided into 5 groups (6 per group): (a) control plasmid (Ctrl); (b) NMI overexpression (NMI); (c) control plasmid + lipopolysaccharides (Ctrl + LPS); (d) NMI overexpression plasmid + LPS (NMI + LPS); (e) NMI shRNA + LPS (shRNA + LPS). As a transfection reagent, the complex of cholesterol-conjugated plasmid (10 μg) suspended in 100 μl saline were injected twice a week for 3 weeks. The dose of LPS for each mouse is 10 μg/kg, and LPS was injected twice a week for 2 weeks. Tumors volumes and body weights were measured every day. Tumor volumes were calculated as V = 1/2 (width2 × length). Mice were humanely sacrificed by euthanasia after treatment.
Human tissue microarray
The human tissue micro arrays were purchased from Outdo Biotech Company (Shanghai, China). 75 cases from 75 lung cancer patients in total were arranged into two tissue array blocks.
Human tissue immunohistochemistry
Slides were deparaffinized, rehydrated and then immersed in a target retrieval solution (pH 6) and boiled at medium baking temperature for three times with 10 min once in a microwave. After blocking the slides with 3% BSA, the sections were incubated with primary antibodies against NMI (Santa Cruz Tech., dilution 1:100) and COX-2 (Epitomics, dilution 1:100), and then with HRP-labeled anti-rabbit IgG secondary antibody. The specimens were counter stained with hematoxylin. A negative control was obtained by replacing the primary antibody with a regular rabbit IgG. The target-positive cells were counted in 3–4 different fields and photographed using an Olympus microscope, and the immunoreactions were evaluated independently by two pathologists blinded to the clinicopathologic information to ensure proper tissue morphology. Antibody staining intensity was categorized: no staining as 0, weak as 1, moderate as 2, and strong as 3. A five-scale system was used to categorize the percentage of cells strained: 0 (no positive cells), 1(<25% positive cells), 2 (25%–50% positive cells), 3 (50%–75% positive cells), and 4 (>75% positive cells). The score for each tissue was calculated by multiplying the intensity index with the percentage scale, and the range of this calculation was therefore 0–12. The median value of NMI scores was employed to determine the cutoff. Tumors with NMI scores lower or equal to the median were designated as “low expression”, whereas those with scores higher than the median were designated as “high expression”.
TCGA Data analysis
The mRNA sequencing data of lung adenocarcinoma samples (n = 576) from The Cancer Genome Atlas (TCGA) were downloaded from UCSC XENA database, gene expression RNAseq (IlluminaHiseq pancan normalized) dataset. All samples were divided into three groups (i.e. high or H, red in heat map; medium or M, black in heat map; and low or L, green in heat map) based on raw gene expression values. NMI scores higher than 66.6% of the patients’ NMI scores were designated as “high expression”, between 66.6% and 33.3% of the patients’ were “medium expression”, and lower than 33.3% of the patients’ were “low expression”. All the samples were selected and used for analysis. The correlations of gene expression were accessed by Pearson correlation coefficient.
Student’s t-tests were used to compare two independent groups of data. Chi-square tests were applied to analyze the correlation between NMI expression and clinicopathologic features of lung adenocarcinomas patients. Survival curves were constructed using the Kaplan-Meier method and were compared using the log rank test. Multivariate Cox proportional hazards analyses used “Enter” modeling to generate models predictive of outcome. Spearman correlation was used to explore the relationship between the abundance of NMI and COX-2. All calculations were performed with IBM SPSS 22 for Windows (SPSS, New York, NY, USA).
The results were presented as the mean ± SD of three independent tests. *P < 0.05, **P < 0.01, significant difference between the treatment and control groups.