Patients and materials
In this retrospective study were included 319 NSCLC patients who had not previously received radiotherapy, chemotherapy, or targeted therapy and who had been diagnosed and treated surgically at the Department of Thoracic Surgery of Tangdu Hospital of Air Force Medical University (Xi’an, China) between May 2009 and January 2014.
The study was approved by the ethics committee of the hospital (ethical approval number 202003–019). Written informed consent was obtained from all patients before any study-related procedures began. The complete follow-up was updated until death or January 2019, whichever came first. Furthermore, 12 pairs of frozen NSCLC tissues and corresponding tumor-adjacent normal tissues were randomly selected for western blot analysis.
Tissue samples of NSCLC patients, tissue microarray, and immunohistochemistry
The 319 pairs of formalin-fixed NSCLC tissues and corresponding tumor-adjacent normal tissues were placed into a paraffin-embedded tissue microarray (TMA). Immunohistochemistry (IHC) staining was carried out on TMA sections according to standard practice, using the primary antibodies of anti-HDAC7 (1:100, #33418, Cell Signaling Technology (CST)) and anti-FGF18 (1:200, 11495–1-AP, Proteintech).
The scoring of the IHC intensity included negative (score 0), weak (score 1), moderate (score 2), or strong staining (score 3). The proportion of positive stains was scored as 0 (< 5%), 1 (6%–25%), 2 (26%–50%), 3 (51%–75%), or 4 (> 75%). These 2 scores were multiplied to produce the total score. The IHC staining was read by 2 expert pathologists. Any disagreement between them was resolved by discussion with a third pathologist. All pathologists were blinded with no information of the clinical data until statistical analysis. IHC staining results with low or high levels of HDAC7 or FGF18 expression were stratified by their respective average score.
Cell culture and lentivirus infection
Human NSCLC cell lines (H1299, A549, Calu-1, SK-LU-1) and HEK-293T were obtained from the American Type Culture Collection (ATCC), and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, supplemented with 10% fetal bovine serum (FBS, Biological Industries), 1% (v/v) penicillin–streptomycin solution (Hyclone). The HDAC7, FGF18, USP10 and respective control lentiviruses were all obtained from Genechem Corporation (Shanghai, China). H1299, A549, Calu-1, and SK-LU-1 cells were performed lentiviral infection according to the protocol of the Genechem Recombinant Lentivirus Operation Manual and further selected for stable cell lines.
Analysis of cell viability
Cell viability was assessed using Cell Counting Kit-8 (CCK-8, 7Sea) following the manufacturer’s instructions. Optical density (OD) values were obtained at 450 nm using a microplate reader (SpectraMax M5, Molecular Device).
5-Ethynyl-2′-deoxyuridine incorporation assay
Cell proliferation was assessed by 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay. BeyoClick EdU Cell proliferation Kit with Alexa Fluor 594 (Beyotime) was used according to manufacturer’s instructions. For confocal microscopy, cells were imaged on the Olympus FV1000 confocal microscope (Olympus). EdU-positive cells were manually counted and expressed as the percentage of cells calculated from nuclear labeling with Hoechst 33342.
Colony formation assay
For clonogenic assays, 500 cells were seeded and cultured in the 6-well plates for 10–14 days. Then, colonies were fixed with formalin and stained with 0.1% crystal violet (Solarbio). After the plates were photographed, colony numbers were counted using the ImageJ software (National Institutes of Health).
Scratch wound assay
Scratch wound assays were performed in 6-well plates. Once cells were 100% confluent, cells were washed once with phosphate-buffered saline (PBS) and cultured in serum-free media (SFM) for 24 h. A wound was created by scraping the cell monolayer with a p200 pipet tip and then media was immediately replaced with SFM. The digital images were taken immediately after scratching and 24 h post scratch with an inverted microscope. The areas of scratch without cells immediately after scratching and the remaining areas without cells at the end of the assay were calculated using ImageJ software.
Transwell cell migration assay
The abilities of cell migration were quantified using transwell assays. Cell transwell chambers were obtained from Corning (Costar 3422). Cells were seeded on the upper chamber and filled with 300 μL DMEM medium without FBS, and the lower chamber was filled with 1000 μL DMEM containing 10% FBS. After 24 h, the migrated cells were fixed by formalin, stained with 0.1% crystal violet (Solarbio), photographed, and counted.
Immunofluorescence
Cultured cells were washed once with PBS and fixed with 4% paraformaldehyde for 20 min. Fixed cells were washed 3 times with PBS for 5 min each time. Cells were then permeabilized with Triton X-100 (9002–93–1, Sigma-Aldrich) in PBS at 4 °C for 10 min and blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature. Primary antibodies were diluted and incubated with the cells at 4 °C overnight. Cells were washed 3 times with PBS and incubated with fluorescent secondary antibodies at room temperature for 1 h. 4′,6-diamidino-2-phenylindole (DAPI) was added for 5 min to visualize the nuclei. The fluorescence was observed and photographed under an Olympus FV1000 confocal microscope.
Cytoplasmic and nuclear protein extraction
The nuclear-cytosolic protein isolation kit (P0027, Beyotime) was obtained for extracting cytosolic and nuclear fractions. Cultured cells were washed twice and collected with 1 mL of PBS. The cell suspension was centrifuged in 4 °C at 90 g for 3 min. After the supernatant fraction was discarded, the sediment was resuspended in reagent A with fresh phenylmethylsulfonyl fluoride (PMSF). Samples were vortexed vigorously for 5 s and placed on ice for 15 min before reagent B was added. The samples were then centrifuged at 14,000 g for 5 min at 4 °C. The supernatant fraction containing the cytosolic proteins was carefully collected and used immediately or stored at − 80 °C. The sediment was resuspended in nuclear protein extraction reagent with PMSF and vortexed vigorously for 30 s and then placed on ice for 2 min. The last 2 steps were repeated for 30 min. The samples were centrifuged at 14,000 g for 10 min at 4 °C. The supernatant fractions containing the nuclear proteins were used immediately or stored at − 80 °C. The concentrations of the extracted proteins were measured using a BCA Protein Assay Kit (23227, Thermo Fisher Scientific).
Western blotting
Western blotting was performed according to the procedures described previously [20], loading 25 μg of total protein lysate per lane. Primary antibodies were diluted at 1:1000 and secondary antibodies were diluted at 1:5000. Antibodies against the following proteins were used for the study: HDAC7 (#33418), USP10 (#8501), β-catenin (#8480), acetyl-β-catenin (Lys49; #9030), phospho-β-catenin (Thr41/Ser45; #9565), phospho-β-catenin (Ser552; #5651), phospho-β-catenin (Ser675; #4176), Cyclin E1 (#4129), E-cadherin (#14472), Slug (#9585), β-actin (#3700), Lamin B1 (#13435), and GAPDH (#5174) (CST). HDAC7(26207–1-AP), CDK1(19532–1-AP), CDK2 (10122–1-AP), CDK4 (11026–1-AP), CDK6 (14052–1-AP), Cyclin A2 (66391–1-Ig), Cyclin B1 (55004–1-AP), Cyclin D1 (60186–1-Ig), Cyclin D3 (26755–1-AP), FGF18 (60341–1-Ig), FGFR3 (66954–1-Ig), Snail (13099–1-AP), vimentin (10366–1-AP), ZEB1 (21544–1-AP), Flag (66008–3-Ig), and ubiquitin (10201–2-AP) were also used (Proteintech Group).
Immunoprecipitation assays
Cultured cells were washed 2 times in PBS and lysed with EBC buffer (50 mM Tris–HCl, pH 7.5, 120 mM NaCl, 0.5% NP-40, and protease inhibitor cocktail). The whole cell lysates were collected using centrifugation at 13,000 g for 10 min at 4℃. The protein concentrations were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific).
For the Flag tag immunoprecipitation assay, 1000 μg whole cell lysates were incubated with 6 μL anti-Flag M2 Affinity Gel (A2220, Sigma Aldrich) at 4 °C for 4 h on a rotating incubator. Then, the beads were washed 4 times with NETN buffer (20 mmol/L Tris, pH 8.0, 100 mmol/L NaCl, 1 mmol/L EDTA, and 0.5% NP-40). After being diluted with SDS loading buffer, the beads were boiled for subsequent western blotting. For endogenous immunoprecipitation assay, precleared lysates were incubated with 8 μL indicated immunoprecipitation antibody overnight at 4 °C, followed by the addition of 40 μL of prewashed PuroProteome Protein G Magnetic Beads (LSKMAGG02, Millipore) for 2 h at 4 °C. Beads were washed 4 times with NETN buffer, mixed with SDS loading buffer, and boiled. The supernatant fractions were then subjected to western blotting according to standard protocols. Normal mouse immunoglobin G (IgG) or rabbit IgG (Millipore) were used as the negative control.
RNA sequencing and pathway enrichment analysis
Total RNA was extracted from control and HDAC7 overexpression groups of SK-LU-1 cells using TRIzol reagent (Invitrogen). Each group was prepared with 3 parallel replicates. Further RNA sequencing (RNA-seq) detection and analysis were conducted using BGISEQ-500 platform (BGI Corporation). The pathway analysis for differentially expressed genes (DEGs) was performed based on the Gene Ontology (GO) database. The Dr. Tom online platform of BGI was used for data analysis (http://report.bgi.com). The raw RNA-seq data has been uploaded on to the NCBI database (accession number: PRJNA786842).
Real-time quantitative polymerase chain reaction
Total RNA was harvested from cells with TRIzol reagent (Invitrogen). Complementary DNA was generated using a Prime Script RT Master Mix (TaKaRa). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with SYBR Premix Ex Taq II (TaKaRa) to detect targeted messenger RNA (mRNA) levels, and data were analyzed with the MxPro software (Stratagene). GAPDH expression was used as an internal control. The primers were obtained from Genechem Corporation. The primer sequence was as follows:
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HDAC7-F: GAAAGAACAGTCCATCCCAACA
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HDAC7-R: GCTTATAGCGCAGCTTCAGG
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c-Myc-F: GGCTCCTGGCAAAAGGTCA
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c-Myc-R: CTGCGTAGTTGTGCTGATGT
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MMP7-F: GAGTGAGCTACAGTGGGAACA
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MMP7-R: CTATGACGCGGGAGTTTAACAT
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GAPDH-F: TGACTTCAACAGCGACACCCA
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GAPDH-R: CACCCTGTTGCTGTAGCCAAA
TOP/FOP flash activity assay
SK-LU-1 and A549 Cells were seeded into 6-well plates for further experiments. TCF-responsive promoter reporter (TOP-flash) or nonresponsive control reporter (FOP-flash) β-catenin firefly luciferase reporter gene constructs, and a pRL-SV40 Renilla luciferase construct were transfected with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc). The pRL-SV40 Renilla luciferase construct was used as the internal control. Later, Dual-Luciferase Reporter Assay System (Promega Corporation) were used to detect the luciferase activity of TOP and FOP. Relative ratio of TOP and FOP luciferase activity was represented as the mean ± standard error of mean after normalizing to the control.
Enzyme-linked immunosorbent assay
The enzyme-linked immunosorbent assays (ELISA) were performed using the ELISA Kit for Human FGF18 (Cloud-clone Corp, Sec907Hu, Wuhan, China). Briefly, NSCLC cells overexpressing or silencing HDAC7 or the control were cultured for 48 h. Cell-free supernatant were harvested and FGF18 concentrations were quantified with the ELISA Kits respectively according to the manufacturer’s instructions.
Flow cytometry
A549 cells synchronized with serum starvation for 48 h and then released back into the cell cycle were collected at the indicated time point. Cells were washed three times with PBS, centrifuged (1200 g) for 10 min, and resuspended in 75% ethanol at 4 °C overnight. Then cells were washed again with PBS, resuspended and stained with propidium iodide (PI) and RNaseA (C1052, Beyotime, China) according to the manufacturer’s instructions. The cell cycle was then analyzed by flow cytometry on a FACSCalibur flow cytometer (BD Biosciences, USA).
Animal experiments
All animal experiments were approved by the Animal Care Committee of Air Force Medical University. Male athymic nude mice (6–8 weeks, 18–20 g) were obtained from the Laboratory Animal Center of the university. Mice were kept under a 12-h light–dark cycle. Temperatures of 65–75 °F (18–23 °C) with 40–60% humidity were also used as housing conditions for the mice.
In vivo tumor xenograft assay and lung metastasis assay
Nude mice were randomly divided into groups (5 mice per group) and received respective treatments. Briefly, for in vivo xenograft assay, different groups of cells (5 × 106 in 200 µL PBS) were separately and subcutaneously inoculated into the nude mice. The body weight of each mice was measured every 3 days for 21 days. Tumors were then excised from the sacrificed mice for additional analysis.
For tail vein metastasis assay, 5 × 106 cells resuspended in 100 µL of PBS were injected in the lateral tail vein of nude mice. After 4 weeks, an IVIS 100 Imaging System (Xenogen) was used for live animal imaging and acquiring photographic images of in vivo metastases of each nude mice. The mice were then sacrificed. Their lungs were dissected and prepared for standard histological examination. The resulting grayscale photographic and pseudo-colored luminescent images were automatically superimposed using IVIS Living Image software (Xenogen).
Statistical analysis
All experiments included were repeated at least 3 times. The quantitative data were compared between groups using the t-test. Categorical data were analyzed using the χ2-test or Fisher’s exact test. The overall survival rates were determined using the Kaplan–Meier method and log-rank test. Univariate or multivariate survival analysis was carried out using the Cox proportional hazards model. A value of P < 0.05 was considered to be significant. SPSS 24.0 software (IBM Corp) was used to analyze the data.