2.1 Bioinformatics assay
BIOGRID web (https://thebiogrid.org/) was used to find the potential biomarker interaction with NAP1L1 and HDGF. UALCAN web (http://ualcan.path.uab.EdU/) was used to analyze the differential protein expression of NAP1L1 in breast cancer based on the analysis of Clinical Proteomic Tumor Analysis Consortium (CPTAC) database.
2.2 Cell culture
Two breast cancer cell lines (MCF-7 and MDA-MB-231) were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). MDA-MB-231 cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) (PAN-Biotech, Aidenbach, Germany). MCF-7 cell lines were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) (PAN-Biotech, Aidenbach, Germany). All cell lines were incubated in a 5% CO2 humidified chamber at 37℃ and supplemented with 10% fetal bovine serum (FBS; PAN-Biotech, Aidenbach, Germany) .
Breast cancer tissue microarray (TMA) was purchased from Shanghai Outdo Biotech (Shanghai Outdo Biotech, Shanghai, China). They were used to evaluate the NAP1L1 protein expression. Consent from the patients and approval from the Ethics Committee of Shanghai Outdo Biotech were obtained before using the clinical samples for research purposes. Tissue sections from the in vivo experiments were used to detect Ki67 and PCNA protein expression levels using immunohistochemistry. The antibodies used were rabbit anti-PCNA (1:200; Proteintech, Rosemont, USA) and mouse anti-Ki67 (1:200; Signalway Antibody, Maryland, USA). The indirect streptavidin-peroxidase method was used according to the manufacturer’s standard experiment guidelines. Cell staining was respectively scored by two pathologists blinded to the clinical parameters. The extent of staining, defined as the percentage of positively stained tumor cells in relation to the whole tissue area, was scored on a scale of 0–4 as follows: 0, < 10%; 1, 10–25%; 2, 26–50%; 3, 50–75%; and 4, > 75%. The staining intensity was scored as 0–3 (Negative: 0; Weak expression: 1; Positive expression: 2; Strong expression: 3. The score represents the product of the positive staining score, and the color intensity score was used as the final staining score for NAP1L1, Ki-67, and PCNA (0–12). For statistical analysis, final staining scores of 0–6 and 8–12 were considered to show low and high expression, respectively.
2.4 RT-PCR and QPCR
Total RNA was isolated from cells. CDNA was synthesized using reverse transcription reagents (TaKaRa Bio, Shiga, Japan), and cDNA was used as a template for amplification using specific primers (Table S1). Bio-Rad T100 and Bio-Rad CFX96 detection systems were applied for RT-PCR and QPCR, respectively, following the manufacturer’s instructions.
2.5 Lentivirus infection
Lentiviral particles carrying the ShRNA-NAP1L1 precursor were provided by GeneChem (Shanghai, China). MCF-7 and MDA-MB-231 cells were infected with the lentiviral vector. The silencing efficiency for NAP1L1 was tested by Western blot analysis.
2.6 SiRNA and plasmid transfection
SiRNAs for NAP1L1 were designed and synthesized by RiboBio (Guangzhou, China). Plasmids for HDGF were obtained from Vigene Biosciences. Twelve hours before transfection, the breast cancer cells were plated into 6-well plates (Nest Biotech, China) and cultured to 30–50% confluence. SiRNAs or plasmids were then transfected at a concentration of 50 nM using the Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.
2.7 MTT assay
The breast cancer cells (2,000/well) were seeded into 96-well plates. For lentivirus-mediated shNAP1L1 expression, the cells were incubated for a week. For transient transfections with si-NAP1L1, the cells were cultured for four days. Subsequently, 20 µL of MTT (5 µg/µL in PBS) (Sigma, St Louis, MO) solution was added to each well and incubated for 4 h. Then, the formazan crystals formed by viable cells were solubilized in 150 mL dimethyl sulfoxide (Sigma, St Louis, MO), and the absorbance (OD) was measured at 490 nm. All the experiments were repeated at least three times.
2.8 Plate clone formation
Clone formation was studied following our previous study. The cells were seeded in 6-well culture plates at 500 cells/well. After incubation for 14 days, the cells were washed twice with D-Hanks solution and stained with hematoxylin solution. The number of colonies was counted under a microscope. All experiments were performed at least three times.
2.9 Edu staining
For the Edu incorporation assay, the proliferating breast cancer cells were examined using a Cell-Light Edu Apollo 488 or 567 In Vitro Imaging Kit (RiboBio) following the manufacturer’s protocol. After incubation with 10 mM Edu for 2 h, the breast cancer cells were fixed with 4% paraformaldehyde, permeabilized in 0.3% Triton X-100, and stained with Apollo fluorescent dyes. A total of 5 mg/mL of DAPI was used to stain the cell nuclei for 10 min. The number of Edu-positive cells was counted under a fluorescent microscope in five random fields. All assays were independently performed three times.
2.10 Subcutaneous tumorigenesis in nude mice
A total of 5×106 logarithmically growing breast cancer cells (MCF-7 and MDA-MB-231) carrying the ShRNA-NAP1L1 and their corresponding control cells were injected into the fourth pair of nude mice breast fat pads (BALB/C, nu/nu, female 3 weeks-old, one group = 5). The animals were fed an autoclaved laboratory rodent diet. On the 24th day, the tumor tissues were excised and weighed. All animal studies were conducted in accordance with the principles and procedures outlined in the Southern Medical University Guide for the Care and Use of Animals.
2.11 Western blot analysis
The extracted proteins were separated by 10% SDS-PAGE and further transferred onto PVDF membranes (Millipore, Bedford). Antibodies including NAP1L1 (1:1000; Proteintech, Rosemont, USA), CCND1 (1:1000; Proteintech, Rosemont, USA), HDGF (1:1000; Proteintech, Rosemont, USA), and c-Jun (1:1000; Proteintech, Rosemont, USA) were used in the Western blot assays based on the manufacturer’s instructions. Detection was performed using the ECL Plus Western blotting detection reagents (Millipore, USA). The specific protein expression levels of the blots were normalized to GAPDH (1:1000; Bioworld, Nanjing, China).
2.12 Coimmunoprecipitation (Co-IP) assay
Co-IP was carried out using the Pierce Co-IP Kit (Thermos Scientific, USA) following the manufacturer’s instructions. The total proteins were extracted and quantified. A total of 3000 specific protein expression levels of the blots were determined using normaanti-NAP1L1 (Abcam), anti-HDGF (Proteintech), anti-c-Jun (Proteintech), and anti-IgG antibodies for 12 h at 4 ℃. The beads were washed, eluted in a sample buffer, and boiled for 10 min at 100 ℃. The immune complexes were subjected to Coomassie Brilliant Blue staining and Western blot analysis. Anti-IgG was used as a negative control.
2.13 Confocal microscopy
The breast cancer cells were cultured overnight (2ver5 /well) before they were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 at room temperature. The cells were incubated with anti-NAP1L1, anti-HDGF, and anti-c-Jun antibodies for 1 h at room temperature. After incubation for half an hour at 37 ℃ with a secondary antibody, coverslips were mounted onto the slides with a mounting solution containing 0.2 mg/mL DAPI. The images were captured by laser scanning confocal microscopy (Zeiss LSM 800).
2.14 Statistical analysis
Statistical analyses were carried out using the SPSS 20.0 statistical software package (SPSS, Chicago, IL, USA). Data are shown as the mean ± SD from at least three independent experiments. Two-tailed Student’s t-test was applied for comparisons between groups. Survival analysis was performed using the Kaplan–Meier method. All statistical tests were two-sided; single, double, and triple asterisks indicate statistical significance (*P < 0.05, **P < 0.01, and ***P < 0.001).