Ethics approval and consent to participate
All animal procedures were supervised and approved by the Jinzhou Medical University Laboratory Animal Welfare and Animal Experimental Ethical Committee.
Study design
Downloaded clinical data of CENP-N mRNA expression on breast cancer and paracancerous tissues were analyzed to compare the differences. In order to confirm the potential role of CENP-N on breast cancer, we examined the malignant phenotype of breast cancer cells with or without deficient expression of CENP-N in vitro or in vivo. Finally, microarray analysis for genome-wide effects of silencing CENP-N in breast cancer MDA-MB-231 was performed to find the changed and associated signaling pathways, and diseases and functions.
Online data and analysis of breast invasive carcinoma (BRCA)
For the TCGA set, clinical data and CENP-N mRNA expression (RNA-seq and RNA-seq V2) were downloaded form the TCGA data portal (http://tcga-data.nci.nih.gov/tcga/). The data included 1094 samples with the CENP-N mRNA expression, of the 106 samples have their paired CENP-N mRNA data in the paracancerous tissues. The differential expression of CENPN between breast cancer and adjacent normal tissues were evaluated. Overall survival analyses between breast cancer patients separated by median of CENPN mRNA expression were performed using the downloaded data and online database (http//:kmplot.com).
Cell culture
MDA-MB-231 and MCF7 human breast cancer cells were purchased from American Type Culture Collection (Manassas, VA, USA), and National Science & Technology Infrastructure (Beijing, China). Cells were maintained in DMEM (Coring, New Jersey, USA) medium containing 10% fetal bovine serum (Gibco, thermo Fisher Scientific, Inc., USA), and 1% penicillin and streptomycin in a 5% CO2 humidified atmosphere. Trypsin-EDTA was used to detach cells.
Lentiviral transfection
105 MDA-MB-231 or 25 × 104 MCF7 cells was seeded in 6-well plates and incubated in 5% CO2 at 37˚C overnight. Afterwards, in order to construct experimental groups, cells was transfected with CENPN-siRNA GFP lentivirus (shCENPN) (Genechem, Shanghai, China) or a control GFP lentiviral vector (shCtrl) (Genechem, Shanghai, China), and the multiplicity of infection (MOI) was 20. 16h later, no obviously cytotoxic effects were observed, and infection solution was replaced by standard cell culture medium. After another 72h, ~80% confluence was reached and cells were observed under fluorescence microscopy and harvested for further qRT-PCR and Western blot to evaluate the transfection efficiency.
Quantitative reverse transscription-PCR (qRT-PCR) analysis
Total RNA was extracted using a GeneJET RNA purification Kit (K0731, ThermoFisehr Scientific, USA) according to manufacturer’s instructions. For each reaction, a total of 2 µg purified RNA was reverse-transcribed into cDNA using the reverse transcription kit (A5001, Promega, Madison, WI, USA). Afterwards, using qRT-PCR Master Mix (A6001, Promega, Madison, WI, USA), mRNA expression of CENPN was evaluated. The primer sequence of Real-time PCR were: CENP-N upstream, 5’-ACAAACCTACCTACGTGGTGT-3’; downstream, 5’- CCAGAAGCGGTGTATTGCG, and GAPDH upstream, 5’-TGACTTCAACAGCGACACCCA-3’; downstream, 5’- CACCCTGTTGCTGTAGCCAAA. Relative quantification was used to analyze mRNA expression of CENP-N.
Western blot assay
After lentiviral transfection, cells were harvested, and proteins were extracted using a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Lysates were centrifuged 12,000g for 30 min at 4˚C, supernatants were collected. Using a BCA protein assay kit (PC0020, Solaria life sciences, China) the protein concentration was determined. A total of 80 μg of protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). PVDF membrane were blocked in 5% non-fat milk for 1 hour, and incubated with primary antibodies against CENP-N (ab57660, abcam, China), STAT2 (#4594, cell signaling technology, China), IRF1 (ab55330, abcam), CCND1 (#2978, cell signaling technology), CASP1 (ab1872, abcam), ISG15 (#2758, cell signaling technology), IFIT1 (#12082, cell signaling technology) and GAPDH (sc-32233, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Subsequently, PVDF membranes were incubated with goat anti-mouse (sc-2005, Santa Cruz) or goat anti-rabbit (sc-2004, Santa Cruz) secondary antibody conjugated with horseradish peroxidase at room temperature for 1 to 2 hours. Protein levels were evaluated on a chemiluminescent imaging system (LAS4010, GE Healthcare, USA) after exposure to electrochemiluminescence reagent (TransGen Biotech, Inc., Beijing, China). Gray values of protein bands were analyzed by Image J software (National Institutes of Health, USA).
Cell counting assay
Cells transfected with lentivirus carried with green fulorescence protein (GFP) can be captured and counted by Celigo Image Cytometer (Nexcelom, USA). After lentiviral transfection, MDA-MB-231 (1500 cells in 100 µl per well) and MCF7 (2000 cells in 100µl per well) cells were seeded into 96 well plates. In the next five days, the number of cells was counted using Celigo Image Cytometer each time a day.
MTT assay
Lentivirus transfected MDA-MB-231 (2×104 cells/ml) and MCF7 (3×104 cells/ml) cells suspension were prepared and seeded into 96 well plates (100 µl per well). After attachment, cells were incubated in 5% CO2 at 37˚C for 1 to 5 days. Next, cells were added with 10 µl MTT reagent (5mg/ml in PBS) and incubated with cells for 4h. In order to dissolve the formazan product, 100 µl of dimethyl sulfoxide (DMSO) was added. At 490 nm, the absorbance was measured using a microplate reader, and continuous 5-day changes of the optical density (OD) value were analyzed.
Apoptosis assay
Cells of MDA-MB-231 and MCF7 were trypsinized, counted and planted in 6-well plates. In the second day, cells were transfected with shCENPN and shCtrl lentivirus. 72 h later, according instruction of Apoptosis detection kit (88-8807, eBioscience, USA), transfected cells reach ~80% confluence, were trypsinzed, washed, and 106 cells were resuspended in 200µl 1× biding buffer. Afterward, 10µl Annexin V-APC staining solution was added and incubated with cell suspension at room temperature avoiding from light for 10-15 min. Finally, 800 µl 1× biding buffer was added and cell apoptotic percentage was examined within 1 h.
Caspase3/7 assay
Cells were transfected with lentivirus as described in apoptosis assay. Afterward, cells were trypsinzed, counted and seeded into each 96 well plate at a density of 10000 cells, and were cultured at 37˚C in a humidified incubator containing 5% CO2 overnight. Caspase3/7 activity was examined by Caspase-Glo®3/7 assay kit (G8901, Promega, USA). According to manufacturer’s instruction, Caspase-Glo solution was prepared. After, each well was added 100 µl Caspase-Glo solution and shaking at a speed of 300-500 rpm using a 96-well plate shaker for 30 min. For another 2 h incubation at room temperature, micoplate reader was used to determine intensity of fluorescence signals. Caspase3/7 activity was evaluated by analyzing the examined data.
Nude mice and in vivo Tumor growth assay
4-week old female BALB/c nude mice averagely weighted 21 gram were obtained from Shanghai Lingchang Biotechnology Co., Ltd. The mice were bred in specific pathogen free housing, and 3-5 female mice were kept in a cage, which was away form light. The bedding material and food were all sterilized and provided by BEIJING HFK BIOSCIENCE CO., LTD. All experiments were conducted under the approval of Jinzhou Medical University Laboratory Animal Welfare and Animal Experimental Ethical Committee.
A total of 20 mice were randomized and allocated to two groups using statistical software (JMP software). MDA-MB-231 cells were transfected with shCENPN and shCtrl lentivirus. Afterwards, 107 transfected MDA-MB-231 cells were inoculated subcutaneously into right axillae of nude mice (10 miles for each group). When tumor could be observed, tumor length and width were measured and tumor volume was calculated as volume =length×width2 /2. The measurement was conducted each three days until tumor volume up to 1cm3. At this time, nude mice were killed by cervical dislocation, and tumor were removed and weighted up. Photos of nude mice and tumor were taken and preserved.
Microarray analysis
Using a GeneChip® PrimeView™Human Gene Expression array (901838, Affymetrix, USA), which can measure gene expression of more than 36,000 transcripts and variants per sample, the genome-wide effects of CENP-N knockdown were evaluated. Total RNA of transfected MDA-MB-231 cells were extracted, and examined for quality control using Thremo Nanodrop 2000 spectrophotometer and Alient Bioanalyzed 2100. Amplified RNA (aRNA) was then prepared using GeneChip® 3' IVT labeling kit (Affymetrix) according to manufactuters’ instructions. Next, GeneChip Hybridization Wash and Stain Kit (Affymetrix) were used for target hybridization and fluidics setup, according to manufacturers’ instructions. Finally, array scanning was performed using CeneChip Scanner 3000 (Affymetrix). Data were collected and normalized to find the differentially expressed gene that the absolute of gene expression fold change is more than 1.5.
Ingenuity Pathway Analysis
Datasets of differentially expressed genes from microarray analysis were imported into the Ingenuity Pathway Analysis (IPA) tools (Ingenuity® Systems, USA). According to manufactures’ instructions, IPA is an integrative online analysis software containing more than 5 millions of biological information from a number of literatures and more than 30 public databases. Different functional modules were set to help understand interaction network among different molecules and diseases and functions. Classical pathway analysis was performed to find the possible activated or inhibited signaling pathways involved in CENP-N knockdown. In the present study, CENP-N associated diseases and functions were also analyzed. During the analytic process, two statistical indicators were used, Z-score was to evaluate which process is activated (Z-score>2) or suppressed (Z-score<-2), as for P value was to assess whether the difference is significant.
Statistical analyses
JMP software (version 11, SAS Institute Co. Ltd, China) was used for statistical analysis. Student’s t test was used to analyze difference between two groups. One-way ANOVA followed by Turkey-Kramer HSD method was used to determine differences among multiple groups. Analysis of variance of repeated measures was conducted to determine differences of tumor growth in continuous 5 days. Kaplan-Meier method was performed to examine differences of overall survival between patients in low-expression CENPN group and high-expression CENPN group. P<0.05 was identified as a statistical significance.