Biological information analysis
GSE43458 (including 80 LUAD and 30 adjacent normal lung tissues) and GSE75037 (including 83 LUAD and 83 adjacent normal lung tissues) data were obtained from the GEO database; the GEO2R method of analysis was performed on the two GEO datasets. In addition, RNA sequencing data from the TCGA LUAD cohort were downloaded, containing 478 LUAD and 57 adjacent normal lung tissues. The “limma” package in R (version 3.6.2; R Foundation for Statistical Computing, Vienna, Austria) was utilized to identify the DEGs between LUAD and normal lung tissue samples; genes—with “adjusted P-value < 0.05” and “|logFC| ≥ 1” as basic parameters—were selected as DEGs from each dataset.
The R package, clusterProfiler [16]—able to analyze and visualize the functional profiles of the genome coordinates—was used to perform GO functional enrichment and KEGG pathway enrichment analysis. PPI analysis was performed using STRING (https://string-db.org/) to identify DEGs; the results were visualized using Cytoscape [17], while hub genes were identified using CytoHubba [18] (degree ≥ 40).
To explore the possible molecular mechanisms of NCAPG in lung cancer cell lines, we downloaded and compiled RNA-seq data from 80 LUAD cell lines from the Cancer Cell Line Encyclopedia website [19]. We performed gene set enrichment analysis (GSEA) [20] on the data of the LUAD cell lines to further explore the signaling pathways of NCAPG.
The Comparative Toxicogenomics Database (CTD, http://ctdbase.org/) [21] is a website that helps improving understanding of the chemical-gene-disease relationship. In CTD, the links between chemical-gene-disease are extracted from the literature. Furthermore, a network between chemotherapeutic drugs and NCAPG was constructed based on CTD and visualized using Cytoscape.
Patients and tissue samples
We collected 292 paraffin-embedded primary LUAD cancer tissues from patients at the Fujian Provincial Hospital, Fujian Medical University. Samples were collected between January 2010 and January 2021. The Ethics Committee of Fujian Provincial Hospital approved the use of all samples (K2021-04-092). Patients with a precise diagnosis and complete clinical information, who had not previously received radiation or chemotherapy, were included.
Immunohistochemical analysis
The samples were prepared with a thickness of 0.5 μm, dewaxed, rehydrated, and incubated with NCAPG antibodies (1:200; Abcam, Cambridge, UK) and Ki67 antibodies (1:200; Abcam) in a wet box at 4 °C. After removing the antibodies, the samples were incubated with HRP-conjugated secondary antibodies; diaminobenzidine (1:50) was then added, and the cells were washed with phosphate-buffered saline (PBS) after 1 min.
Five fields of view were randomly selected for each slice and scored using a double-blind method by more than two experienced pathologists; the scores were based on the percentage of stained cells and degree of staining. Regarding the number of pigmented cells, 0 indicated that 0–10% of cells were stained, and 1, 2, 3, and 4 respectively indicated that 11–25%, 26–50%, 51–75%, and > 75% of cells were stained. Regarding the degree of staining, 0 indicated no staining, and 1, 2, and 3 respectively indicated light yellow, yellow, and brownish yellow staining. The two scores were multiplied to obtain a final quantification for each sample. Staining intensity was graded as negative for scores 0–3, weakly positive for scores 4–6, moderately positive for scores 7–9, and strongly positive for scores 10–12. Negative and weakly positive scores were considered to indicate low expression; conversely, moderately and strongly positive scores were considered to indicate high expression.
Cell culture and infection
The human bronchial epithelial and LUAD cell lines—BEAS-2B, and PC9, A549, H827, H1299, and H1975—were purchased from the cell bank of the Chinese Academy of Sciences in Shanghai, China; they were then cultured in RMPI-1640 medium with 10% fetal bovine serum and incubated at 37 °C with 5% CO2. Cell lines were authenticated to be free of mycoplasma contamination. PC9 and A549 cells were infected with lentiviruses harboring NCAPG overexpression. NCAPG-targeting short hairpin RNA (shRNA) was synthesized by Hanheng Biology Co., Ltd. (Shanghai, China). The target sequences of shRNA were as follows: sh-NCAPG 1#: 5′-GCTGTCAGAAAGCTGGCTTAT-3′ and sh-NCAPG 2#: 5′-GCTGAAACATTGCAGAAATGT-3′. RepSox was purchased from Selleck (Shanghai, China).
Western blot assay
The tissues and cells were lysed with a protease inhibitor cocktail for 20 min and then centrifuged to remove the supernatant. Protein concentration was quantified using a BCA kit; 30 μg of lysate from each sample was then separated by SDS–polyacrylamide gel electrophoresis, then blotted onto polyvinylidene fluoride membranes. Next, the primary antibody was added after sealing with 3% bovine serum albumin. Detailed information regarding the antibodies used in this study is listed in Additional file 1: Table S1. After washing the primary antibody with PBS, the corresponding HRP-linked secondary antibody was added; enhanced chemiluminescence was added for detection.
Cell Counting Kit 8 (CCK8) assay
CCK8 (Beyotime Biotechnology, Shanghai, China) was used to evaluate cell proliferation. After lentivirus infection, CCK8 reagent was added to the culture medium; the absorbance at 450 nm was measured after 24, 48, 72, and 96 h.
Clone formation experiment
Cells were trypsinized and inoculated in a six-well plate at approximately 1000 cells/well. The plate was frequently observed, and the culture was stopped when clones were visible; the plate was then fixed with methanol, stained with crystal violet, and quantified.
Wound healing assay
Cells were cultured in a six-well plate at a cell density of 5 × 105 cells/well. When the cells reached 90% confluence, a straight line was vertically drawn at the bottom of the well using the tip of a 10 μL pipette. Debris was removed and cultured in serum-free RPMI-1640 medium, and photographs were acquired at 0 and 48 h.
Transwell migration assay
In the cell migration experiment, 200 μL of cell suspension (1 × 105/mL) with serum-free RPMI-1640 medium was added to the upper well, while 600 μL medium containing 20% fetal bovine serum without cells was added to the lower chamber. After 24 h, the upper chamber was carefully removed, washed, fixed with paraformaldehyde, and stained with crystal violet. The non-migrating cells in the upper chamber were then gently wiped off with a cotton swab; photographs were subsequently obtained. Five fields of view were randomly selected for counting.
In the invasion experiment, the upper chamber was precoated with Matrigel (BD Biosciences, San Jose, CA) before the cells were added; subsequent steps were consistent with those of the migration experiments.
Xenograft tumor model
Four- to five-week-old male BABL/c nude mice (14–18 g) were purchased from Slack Laboratory Animal Co., Ltd. (Shanghai, China). After lentivirus infection, PC9 cells (5 × 106)—including sh-Control, sh-NCAPG 1#, and sh-NCAPG 2#—were injected into the right flanks of mice. Tumors were measured every 7 days, and their volumes were calculated as follows: (length × width2)/2. On day 28, the tumors were harvested for immunohistochemical analyses.
Statistical analysis
Statistical analyses were performed using SPSS 22.0 (IBM Corp., Armonk, NY). The association between gene expression and clinicopathological characteristics was analyzed using the chi-squared test, while the Kaplan–Meier method was used for the survival analysis. In addition, differences between two groups were analyzed using Student's t-test, while differences among three or more groups were analyzed using one-way analysis of variance; statistical significance was set at p < 0.05. The assays were performed independently at least three times.
