Cell lines and cell transfection
A549 and NCI-H1299 cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences and NCI-H1299 were cultured in RPMI-1640 medium (Gibco) with 10% FBS and A549 was maintained in McCoy’s 5A Medium with 10% FBS. All cells were cultured in a humidified cell culture incubator at 37 °C under 5% CO2 with culture medium changed every 72 h.
For stable gene expressing, lipofectamine RNAimax (Cat. #13778075, Thermo fish) were used for cell A549 and NCI-H1299 transfection with lentiviral plasmids collected. Cells were harvested after 72 h culturing, and cell infection efficiency was valued with LV-shCtrl cells as control.
Immunohistochemical (ihc) Staining
Human lung cancer and para-normal tissue chip (Cat. #HLugA180Su05, Shanghai Outdo Biotech Company) was used and patients’ information was collected. For IHC staining, deparaffinized and rehydrated tissue sections were blocked and incubated with primary antibody ZNF280A (Cat. #bs-12839R, BIOSS) and followed incubated by secondary antibody. DAB color was developed with diaminobenzene and hematoxylin. Slides were pictured with microscopic and viewed with ImageScope and CaseViewer. All slides were examined randomly by two independent pathologists and IHC outcomes were determined by staining percentage and intensity scores. Staining percentage scores were classified as: 1 (1%-24%), 2 (25%-49%), 3 (50%-74%) and 4 (75%-100%). Staining intensity were scored 0 (Signalless color) to 3 (light yellow, brown and dark brown). Antibodies used in IHC were listed in Table S1.
Rna Interference And Plasmids Packaging
shRNA sequences targeting human ZNF280A and EIF3C gene were designed and cDNAs were synthesized by Shanghai Yibeirui Bioscienceres, Co., Ltd. and subsequently cloned into luciferase-labelled BR-V-108 vector. In addition, ZNF280A was amplified and cloned into the BR-V112 vector after double digestion by BamHI and AgeI, and sequenced. Lentiviral particles were collected, following co-transfection using pHelper 1.0 and pHelper 2.0 vector for plasmids packaging. The sequences used were listed in Table S2.
Rna Extraction And Rt-qpcr
After 72 h for ZNF280A and/or EIF3C RNA expressing, A549 and NCI-H1299 cells in triplicate were fully lysed and total RNA was extracted using TRIzol reagent (Sigma). The RNA quality was evaluated by Nanodrop 2000/2000C spectrophotometer (Thermo Fisher Scientific). cDNA was reversely transcribed from RNA using Promega M-MLV Kit (Promega) and qPCR was performed with SYBR Green mastermixs Kit (Vazyme) by applying Biosystems 7500 Sequence Detection system. GAPDH was acted as inner control, and the primers used for the PCR reaction were showed in Table S3. The relative quantitative analysis in gene expression data were analyzed by the 2−ΔΔCt method.
Western blotting (WB), co-immunoprecipitation (Co-IP) and Human Apoptosis Antibody Array
Cells were lysed in ice-cold RIPA buffer (Millipore), and the protein were collected and the concentration was detected by a BCA Protein Assay Kit (HyClone-Pierce). Protein samples (20 µg per lane) were separated by 10% SDS-PAGE (Invitrogen), and transferred onto PVDF membranes at 4 °C. The membranes were blocked with TBST solution of 5% degreased milk at room temperature for 1 h and incubated with primary antibodies and GAPDH antibodies at 4 °C overnight. Then the membranes were incubated with secondary antibody HRP goat anti-rabbit IgG for 2 h at room temperature. The blots were visualized by enhanced chemiluminescence (ECL) (Amersham).
For Co-IP, prepared proteins were immunoprecipitated by anti-ZNF280A, EIF3C or GAPDH antibody and then subjected to WB with antibody to ZNF280A and EIF3C and related secondary antibodies.
For Human Apoptosis Antibody Array, briefly, 20 µg total proteins were cultured with the antibody-coated array membranes and then continuing incubated with HRP linked Streptavidin conjugate.
All the antibodies used in western blotting were listed in Table S1.
Cell Proliferation Analysis
The cell viability was determined by MTT assay, briefly, transfected A549 and NCI-H1299 cells were stained with MTT reagent (5 mg/mL, GenView) and Formazan was dissolved by DMSO solution. The absorbance values at 490 nm were measured by microplate reader (Tecan) and the reference wavelength was 570 nm.
Cell proliferation rate was analyzed by Celigo cell counting assay. Briefly, targeting cells were seeded at a 96-well plate with 2,000 cells per well. The plate was continuously detected by Celigo (Nexcelom) for 5 days at the same time.
For colony formation assay, cells in the logarithmic growth phase were seeded into 6-well plates in triplicate and further cultured for 8 days. Cell clones were fixed with 4% paraformaldehyde and stained with Giemsa. Then clones were photographed under a fluorescence microscope (Olympus) and colony number (clone contains more than 50 cells) was counted.
Cell Apoptosis And Cells Cycle Assay
The flow cytometric methods of identifying apoptotic cells was applied using Annexin V-APC Apoptosis kit (Cat. #88-8007, eBioscience). For cells cycle assay, cells were stained with 1 mL PI staining solution (40 × PI, 2 mg/mL: 100 × RNase, 10 mg/mL: 1 × PBS = 25:10:1000). FACScan and FlowJo 7.6.1 (Ashland) was used for analyze. Cell apoptosis was measured and the percentage of the cells in G0-G1, S, and G2-M phase were counted and compared.
Cell Migration Assays
In order to analysis the migration ability of transfected cells in our research, wound healing assay and Transwell assay were performed. For wound healing assay, lentivirus transfected A549 and NCI-H1299 cells (5 × 104 cells/well) were plated into 96-well plates for culturing. Scratches were made by a 96 wounding replicator (VP scientific). Photographs were taken by a fluorescence microscope at 0 h, 8 h and 24 h and cell locations were recorded, respectively. Cell migration rates of each cell group were calculated. In transwell assay, cells were seeded into a 24-well plate in the upper chambers, and medium supplemented with 30% FBS was added into in the lower chambers. Cells were fixed with 4% formaldehyde and stained by Giemsa and the migration ability of cells was analyzed.
High-throughput Rna Sequencing
Total RNA from NCI-H1299-shCtrl and NCI-H1299-shZNF280A cells was extracted using TRIzol. RNA quantity and quality were assessed with a Thermo Nanodrop 2000 (1.7 < A260/A280 < 2.2, Thermo Fisher Scientific). Affymetrix PrimeView Human Gene Expression Arrays (Thermo Fisher Scientific) were used for microarray analysis to obtain gene expression profiles according to the manufacturer’s instructions. Significantly differentially expressed genes were selected based on P < 0.05 and |Fold Change| > 1.3. KEGG pathway enrichment analysis was performed for all significant differentially expressed genes.
All animal studies were approved by Ethics committee of Peking Union Medical College Hospital. Female BALB/c nude mice were purchased from Shanghai Lingchang Experimental Animals Co., Ltd. For tumorigenicity, 5 × 106 lentivirus (shCtrl or shZNF280A) transfected NCI-H1299 cells were subcutaneously injected into each mouse (4-week-old, n = 10 per group). Mice’s weight and tumor sizes were recorded 2 times per week and the volume of tumor were calculated as π/6 × L × W2 (W, width at the widest point; L, perpendicular width). Finally, the tumor burden was assessed by bioluminescence imaging with non-invasive IVIS Spectrum Imaging System (Perkin Elmer). Mice were sacrificed then tumors were extracted and imaged.
Ki67 Immunostaining Assay
Mice tumor sections were fixed in 4% paraformaldehyde. Paraffin embedded 5 µm sections were made for H&E and IHC staining. We added citric acid buffer for antigen retrieval at 120 °C. Sections were blocked using PBS-H2O2 with 0.1% Tween 20. Ki-67 antibody was added for incubating at 4 °C overnight and then secondary antibodies were added as well. DAB color was developed with diaminobenzene and hematoxylin. Stained slides were pictured with a microscopic.
Each experiment was repeated three times and the data was shown as mean ± SD. Categorical variables were expressed as percentages. The significance between groups was determined using the two-tailed Student’s t test or one-way ANOVA analysis. Relationship between ZNF280A expression and tumor characteristics in lung cancer patients with was analyzed using Mann-Whitney U analysis and Spearman grade correlation analysis. Statistical significance was calculated by SPSS 22.0 (IBM) and P value < 0.05 was considered statistically significant. Graphs were made using GraphPad Prism 6.01 (Graphpad Software).