Expression and survival analysis
The GEPIA database is an online tumor database integrated with RNA sequencing expressions of 9,736 tumor samples and 8,587 normal samples from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases[20]. The expression of NCAPG in OC and normal tissues were analysed in and visually displayed as boxplot format in GEPIA database (http://gepia.cancer-pku.cn/), log2 (TPM+1) was used for log-scale.
Kaplan Meier-plotter (KM plotter) is another cancer database, but different from the GEPIA database,which based on TCGA, EGA, and GEO (Affymetrix microarrays only) database[21]. The overall survival (OS) and progression-free survival (PFS) of the NCAPG-low and NCAPG-high expression subgroups were compared in KM plotter (http://kmplot.com/analysis/). The hazard ratio (HR) with the 95% confidence interval and the log-rank P-value were calculated and displayed on the plot, and the number-at-risk is displayed below the curves. logrank P-value <0.05 was considered statistically significant.
Tissue microarray
A tissue microarray harboring seventy ovarian cancer tissues and ten para-carcinoma tissues was purchased from bioaitech(Xian, China). Seventy cases of ovarian cancer and ten cases of para-carcinoma tissues were randomly obtained from the national tertiary hospital (China) between 2010 and 2018. According to WHO histology grading: there were 8 cases in grade I, 2 cases in grade I~II, 2 cases in grade II, 2 cases in grade II~III, and 56 cases of grade III. Patients with preoperative radiotherapy or chemotherapy were excluded. OC and para-carcinoma tissues were collected and stored at –80 °C until analysis. Informed consent was obtained from patients prior to specimen collection. The study protocol was approved by the Ethics Committee of Tongxu People's Hospital of Henan Province and all investigations were conducted according to the Declaration of Helsinki.
Immunohistochemistry
An immunohistochemistry was utilized to explore the expression patterns of NCAPG in 70 OC tissues and 10 para-carcinoma tissues. The tissue microarray was drying with drying apparatus AKT-7 (Atobo, Hubei, China), and then dewaxing with xylene (MXB biotechnologies, Fuzhou, China) and hydrating in gradient alcohol(MXB biotechnologies, Fuzhou, China), 3% hydrogen peroxide(MXB biotechnologies, Fuzhou, China) was used to inhibit endogenous peroxidase. The sections were incubated with primary antibody solution against NCAPG (1:20, abclonal, Tokyo, Japan) for 2 hours. After washing for three times, they were covered with HRP anti-rabbit immunoglobulin G (IgG; MXB biotechnologies, Fuzhou, China) for 30 mins. After DAB(MXB biotechnologies, Fuzhou, China) staining and hematoxylin(MXB biotechnologies, Fuzhou, China)counterstaining, the sections were evaluated by two independent pathologists according to staining area and intensity. The scoring criteria for staining intensity were regarded as: 0, no staining; 1, light yellow staining; 2, light brown staining; and 3, dark brown staining. The percentage of stained cells: 1, 0-25%; 2, 26-50%; 3, 51-75% and 4, 76–100%. The product of the two values was defined as the NCAPG staining scores.
Cell Culture
Human ovarian cancer cell lines SKOV3 and OVCAR3 were obtained from Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China) and iCell Bioscience (Shanghai, China), respectively. The human ovarian epithelial cell line IOSE-80 was bought from iCell Bioscience (Shanghai, China). All cell lines were cultured in RPMI-1640 medium (Hyclon,Logan, Utah, USA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% antibiotic/antimycotic solution (Hyclon,Logan, Utah, USA) and placed in a 5% CO2 incubator at 37°C.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cultured cells with Trizol reagent (Vazyme, Nanjing, China) and then reverse-transcribed to complementary DNA (cDNA) using a PrimeScriptTM RT Reagent kit (Vazyme, Nanjing, China). qRT-PCR was performed with a ABI7300 real-time quantitative PCR instrument (Applied biosystems, Foster City, CA, USA) using SYBR Green Master Mix as described by the manufacturer's protocol(TaKaRa, Dalian, China). The primers were synthesized by Generay (Shanghai, China) and the primer sequences used were as follows: NCAPG, forward, 5′- CGCTTTCACGACTTCAGGAT-3’ and reverse, 5′-ATAACACTGCCCGTCTAACTTCT-3′;β-actin-forward,5ʹ-CGTGCGTGACATCAAAGAGAAG -3ʹ and reverse, 5ʹ- CCAAGAAGGAAGGCTGGAAAA -3ʹ. β-actin was used as an internal control. The results were analyzed using 2-ΔΔCt method. All RT reactions were performed in triplicate
Transfection of SKOV3 and OVCAR3 cells with NCAPG-shRNA plasmid
SKOV3 and OVCAR3 cells were cultured in 24-well plates to a confluence of 60–70%, and then transfected with shRNAs targeting human NCAPG using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. Three different shRNAs were used to silence NCAPG: shNCAPG-1, shNCAPG-2, shNCAPG-3. The NCAPG or negative control (NC) plasmid, were both purchased from GeneChem (Shanghai, China). After 48h of transfection, the knockdown efficiency of the shRNAs was confirmed by qRT-PCR.
Cell viability assay
The Cell Counting Kit-8 (CCK-8) assay was performed to investigate the influence of NCAPG knockdown on SKOV3 and OVCAR3 viability. In brief, after culturing and transfecting SKOV3 and OVCAR3 cells as mentioned ahead, 5 × 103 cells were seeded per well in a 96-well plate. CCK8 reagent was added at 0, 24, 48, 72, 96 and 120h, and cell viability was read with a plate reader (ELX800, BiTek, USA) at 490 nm. Three replicates of the experiments were performed.
Colony formation assay
Colony formation assays were performed to evaluate the effect of NCAPG knockdown on OC cell anchorage-independent growth. Briefly, the transfected cells were seeded into 6-well plates at a density of 500 cells/well and cultured for 2 weeks. The culture medium was replaced every 3 days. The colonies were fixed with 4% paraformaldehyde(Solarbio, Beijing, China) and stained with 1% crystal violet(Solarbio, Beijing, China). The number of colonies was counted under a microscope (Olympus CX-23; Olympus Corporation, Tokyo, Japan).
Cell invasion assays
The invasive abilities were evaluated using 8-μm transwell inserts with Matrigel precoating (BD Biosciences, San Jose, CA, USA), Briefly, the transfected cells (1×104 cells/well) were suspended in serum-free medium and placed in the upper chamber, culture medium containing 10% FBS was added to the bottom chamber. Following 24h incubation, the cells that had invaded through the membrane to the lower surface were fixed, stained. Penetrated cells in three randomly selected fields were counted under an inverted microscope (Olympus CX-23; Olympus Corporation, Tokyo, Japan).
Apoptosis and cell cycle analysis
When transfected SKOV3 and OVCAR3 cells grew to 80% confluence, the cells in suspension were collected, and the cells on the dish were digested into single cells suspension by trypsin (Hyclon,Logan, Utah, USA). All cells were collected and mixed with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) solution (KeyGEN, Nanjing, China) for 15 mins at room temperature in the dark. Apoptosis rates were determined by BD flow cytometer (Franklin Lakes, NJ, USA). B3 quadrant represented viable cells and B2 and B4 quadrants represented apoptotic cells. For detection of cell cycle distribution, the transfected cells were digested, washed and incubated with 1μl RedNucleus I (Sysmex Corporation, Kobe, Japan) at 37°C for 10 mins and then measured the cell cycle distribution with BD flow cytometer (Franklin Lakes, NJ, USA)
Western blot analysis
The transfected cells were collected and lysed in RIPA buffer (Solarbio, Beijing, China) mixed with 1% protease inhibitor PMSF(Solarbio, Beijing, China) and incubated on ice for 30 mins. The lysates were centrifuged at 14,000 g for 10 mins at 4°C, and the supernatant was collected. The extracted proteins were separated by 10% SDS-PAGE (Solarbio, Beijing, China) and transferred onto PVDF membranes (Solarbio, Beijing, China). After blocking in 5% BSA, the membranes were incubated with primary antibodies against NCAPG(1:2000, abclonal, Tokyo, Japan), P38MAPK(1:2000, proteintech, Chicago, USA), P53 (1:2000, Wanleibio, Shenyang, China), cyclinD1(1:2000,Cell Signaling Technology, Massachusetts, the United States) and β-actin (1:4000,proteintech,Chicago, USA) at 4°C overnight. After washing with PBST the following day, the membranes were incubated with secondary antibodies (1:5000, proteintech, Chicago, USA) at room temperature for 1h. Finally, the blots were detected by enhanced chemiluminescence (Millipore, Massachusetts, USA). Quantification of the bands was measured with ImageJ (V 1.80).
Statistical analysis
Data were presented as mean ± standard deviation (SD) of at least three independent experiments and analyzed with GraphPad Prism 7 software (GraphPad, San Diego, CA, USA). Kaplan-Meier survival curves and the log rank tests were used to assess significant differences. Differences between groups were analyzed by Student’s t-test for two groups and one-way ANOVA for more than two groups. P values <0.05 were considered statistically significant.