Patients and tissues
In total, 70 patients with operable NSCLC were collected from our previously constructed database . Among them, 37 patients developed brain metastases as the first relapse event within 3 years after surgery, and 5 patients underwent brain metastatic tumor resection. Their formalin-fixed and paraffin-embedded (FFPE) tissues were obtained from the Tissue Bank of Zhejiang Cancer Hospital. The follow-up proceeded as previously described . The median follow-up time was 42 (range 5–108) months. During follow-up, 2 of 33 3-year recurrence-free patients relapsed, 30 of 70 patients died of tumor progression, and 1 of 70 patients was lost to follow-up. All patients provided informed consent before surgery, and the study was approved by the Institutional Review Committee of Zhejiang Cancer Hospital.
Based on previous publications [18,19,20], we selected 36 genes hypothesized to be associated with brain metastasis. These genes represented three functional categories: brain growth and metabolism, epithelial–mesenchymal transition and cytokines. Six 10-μm-thick paraffin sections were cut from each NSCLC FFPE sample for the Luminex assay. Tissue homogenates were prepared using a QuantiGene Sample Processing Kit for FFPE Tissues (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol. A QuantiGene plex 2.0 Reagent System (Affymetrix) was used to capture target RNA and amplify the signal according to the manufacturer’s recommended procedure. Finally, the signal was detected using a Luminex 200 System (Luminex, Austin, TX, USA).
Four-micrometre-thick paraffin sections were cut, mounted on slides, deparaffinized in xylene, rehydrated in decreasing ethanol dilutions and incubated in 3% hydrogen peroxide buffer for 30 min. After blocking of endogenous peroxidase activity, the slides were boiled with citrate buffer (pH 6.0) in a pressure cooker for 90 s, followed by blocking of non-specific binding sites with 5% blocking serum for 30 min at room temperature. The sections were then incubated with rabbit anti-GAP43 antibody (Huabio, Hangzhou, China) at a dilution of 1:1200 overnight at 4 °C. Afterwards, 1:2000 diluted goat anti-rabbit secondary antibody (Dako, Glostrup, Denmark) was applied for 30 min. The reaction products were visualized using 3,3′-diaminobenzidine (DAB; Dako), and the sections were lightly counterstained with hematoxylin and mounted. The results were evaluated by two experienced pathologists and photographed.
The human NSCLC cell lines SK-MES-1, NCI-H1650, NCI-H1975, NCI-H2122, A549, NCI-H838, NCI-H460 and NCI-H661 were purchased from American Type Culture Collection (Manassas, VA, USA). All cells were authenticated by short tandem repeat (STR) profiling and were found to be negative for mycoplasma. SK-MES-1 cells were maintained in Eagle’s minimum essential medium (EMEM; Genom, Hangzhou, China) containing 10% fetal bovine serum (FBS, Gibco); all other cell lines were maintained in RPMI 1640 medium (Gibco) containing 10% FBS. All cells were cultured at 37 °C in a 5% CO2 incubator.
Adherent cells were collected by gentle scraping, washed three times in phosphate-buffered saline (PBS), and lysed in Nonidet P 40 cell lysis buffer supplemented with phosphatase inhibitor cocktail (Cwbio, Beijing, China) and protease inhibitor cocktail (Roche, Mannheim, Germany). A Bradford calorimetric assay (Cwbio) was used to measure the protein concentration of the cell lysates. Thirty micrograms of total protein was separated on a 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). Each membrane was blocked in TBS-Tween-20 (TBS-T) containing 5% non-fat milk at room temperature for 2 h, followed by incubation with anti-GAP43 antibody (1:1000, Huabio, Hangzhou, China) at 4 °C overnight. The next day, the membrane was incubated with an HRP-conjugated secondary antibody (Servicebio, Wuhan, China) at room temperature for 2 h, and the results were detected using an enhanced chemiluminescence (ECL) reagent (Cwbio). Tubulin (1:1000; Antibody Revolution, San Diego, CA, USA) was used as the loading control.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from cells using the TRIzol method. The RNA concentration was measured using a microvolume spectrophotometer, and 500 nanograms of RNA was reverse transcribed using a PrimeScript™ RT Reagent Kit (Takara, Dalian, China). Quantitative PCR (qPCR) was carried out as previously described . The primer sequences were as follows: TTCTTGGTGTTGTTATGGCAAG (GAP43 forward), GAGGAAAGTGGACTCCCACAG (GAP43 reverse), GAAGGTGAAGGTCGGAGTC (GAPDH forward), and GAAGATGGTGATGGGATTTC (GAPDH reverse). The expression levels of GAP43 were analyzed using the 2(−Delta Delta C(T)) method  and adjusted according to the expression levels of the housekeeping gene GAPDH.
GAP43 knockdown and overexpression
For transient knockdown, 4 specific siRNAs targeting GAP43 and a negative control siRNA were designed and inserted into a pRNAT-U6.1/Neo vector. For transient overexpression, GAP43 cDNA was subcloned into a p3 × FLAG-CMV-10 vector, and an empty p3 × FLAG-CMV-10 vector was used as the control. Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA, USA) was used to carry out the transfections, and after 48 h, the cells were harvested to validate the alterations in GAP43 levels or for subsequent experiments.
For stable GAP43 knockdown, a lentiviral vector was applied. Briefly, the GAP43 knockdown lentiviral vector pHBLV-CMV-shGAP43-3flag-EF1-Luc-T2A-Puro and a negative control lentiviral vector were purchased from Hanbio Biotechnology Co., Ltd., Shanghai, China. 293T cells were co-transfected with lentiviral vectors and the helper plasmids pSPAX2 and pMD2G. After 48 h of transfection, the supernatant was collected and purified by centrifugation followed by ultracentrifugation. NCI-H661 cells at 50% confluence were infected with GAP43 knockdown or negative control lentiviral constructs in 8 µg/ml polybrene. Stably transfected cells were selected using 1 µg/ml puromycin for 2 weeks and maintained in 0.5 µg/ml puromycin. GAP43 knockdown was confirmed by western blotting.
Wound healing assay
The wound healing assay was conducted using a 2-Well Culture-Insert (Ibidi, Munich, Germany). Cells were seeded into the wells, and after removal of the insert, a 500-μm cell-free gap was formed. Multiple time-lapse images of the gap were taken at a magnification of 100×.
Transwell migration and invasion assays
Twenty-four-well, 8-μm pore size transwell inserts (Corning, Oneonta, NY, USA) were used. For invasion assays, the inserts were coated with Matrigel (BD, San Jose, CA, USA) before the cells were added. A total of 500 μl of complete medium was added to the lower chamber, and 200 μl of cell suspension (3.0 × 104 NCI-H661 cells for migration, 2.5 × 104 NCI-H661 cells for invasion, 2.0 × 104 NCI-H1650 cells for migration and invasion) in RPMI 1640 supplemented with 1% FBS was added to the upper chamber. After incubation for 24 h, the cells in the upper chamber were removed by gentle wiping using cotton buds, and the cells on the underside of the inserts were fixed with methanol, stained with 0.1% crystal violet, photographed and counted.
In vivo metastasis assay
BALB/c (nu/nu) male nude mice 4–6 weeks of age purchased from Shanghai Slac Laboratory Animal Co. Ltd. were used, each group with 10 mice. Two hundred microliters of PBS containing 1.0 × 106 cells was injected into the left ventricle of each mouse. Metastatic lesions were monitored every week using bioluminescence imaging (BLI). Briefly, the mice were anaesthetized and injected intraperitoneally with 150 µg of d-luciferin (Yeasen, Shanghai, China) per gram of weight. 10 min later, the bioluminescence was imaged using an IVIS imaging system (Caliper Life Sciences, Alameda, CA, USA) and analyzed using Living Image Software 4.3.1.
Adherent cells at 80% confluence on an 8-well glass slide (Millipore) were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 5 min. Next, 100 µl of phalloidin conjugate working solution (Abcam, Cambridge, MA, USA) was added to each well of the slide and incubated with the cells for 60 min at room temperature away from light. The cell nuclei were stained with DAPI (Biosharp, Hefei, China) for 10 min at room temperature away from light, and fluoroshield mounting medium (Abcam) was used to seal the slide. Fluorescence was photographed using a confocal microscope.
F-actin/G-actin ratio in vivo assay
Proteins were extracted and analyzed using an F-actin/G-actin in vivo assay kit (BK037; Cytoskeleton, Denver, CO, USA) based on the manufacturer’s instructions. Briefly, cells were lysed in a detergent-based buffer that dissolved G-actin but not F-actin. A centrifugation step pelleted F-actin while leaving G-actin in the supernatant. Actin in the pellet and supernatant was quantitated by western blotting. The grayscale value of each band was measured using ImageJ software.
Rac1 activation assay
A Rac1 activation assay kit (STA-405; Cell Biolabs, San Diego, CA, USA) was applied to prepare protein according to the manufacturer’s instructions. Briefly, protein was extracted from cells and divided into two equal parts. One part was used to quantitate total Rac1. The other part was incubated with the p21-binding domain (PBD) of p21-activated protein kinase (PAK) Agarose beads to specifically bind and pull-down active Rac1. Next, total Rac1 and active Rac1 were detected by western blotting.
The expression index of each gene from the Luminex assay was averaged in two groups according to the median and then analyzed as categorical variables. The Cox proportional hazards regression model was applied to evaluate the brain metastasis-prognostic value of each gene. The Kaplan–Meier estimator was used to generate brain metastasis-free survival curves, and a log-rank test was used to determine significant differences. A web application called KM plotter (http://kmplot.com/analysis/index.php?p=service&cancer=lung) was employed to obtain the progression-free survival and overall survival curves in public database of lung cancer. Correlations between GAP43 expression and clinicopathological factors were examined using a Chi square test and Mann–Whitney U test. Every experiment was repeated at least 3 times. The results of qPCR, western blotting and cell migration and invasion assays were analyzed using Student’s t-test, and the BLI results were analyzed with a Mann–Whitney U test. IBM SPSS Statistics 24.0 software was used for data processing. A P value < 0.05 was considered statistically significant.