Breast carcinoma datasets
The breast cancer gene expression profiles GSE29431 and GSE42568 were obtained from the publicly available Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). The GSE29431 dataset contained 68 samples, including 12 normal tissues and 54 tumor tissues obtained from BC patients. GSE42568 dataset contained 17 normal tissues and 109 tumor tissues. Furthermore, the gene expression profiling of 1218 BC patients and clinical information were selected from TCGA (http://tcga-data.nci.nih.gov) . Then sort out the matched clinical and expression information for further research.
ONCOMINE database analysis
The transcriptional levels of TRIP13 in BC was examined in the Oncomine database (https://www.oncomine.org) [12], a publicly accessible cancer microarray database, was used to investigate the transcriptional levels of TRIP13 in BC. Fold changes and a p-value of 0.05 was considered significant for the comparison of cancer and normal.
UALCAN analysis
UALCAN (http://ualcan.path.uab.edu) includes TCGA RNA-seq datasets and clinical data from 31 cancer types, which allow us to analyze the relationship between the gene expression and clinical characteristics[13]. Here, we evaluated the relationship between TRIP13 and clinical characteristics including cancer stage, pathological type and other criteria through UALCAN database.
Prognostic survival analysis
The Kaplan-Meier plotter (http://kmplot.com/analysis), an public online tool that can be used to assess the impact of 54675 genes on survival in several cancer types including breast cancer, ovarian cancer, liver and gastric cancer[14]. Prognostic value analysis including the overall survival (OS), relapse free survival (RFS), post progression survival (PPS) and distant metastasis free survival (DMFS) were performed using the Kaplan-Meier method. p-value < 0.05 was considered statistically significant.
LinkedOmics analysis (functional enrichment analysis)
The LinkedOmics database (http://www.linkedomics.org) is a web-based data-mining platform for analyzing TCGA cancer datasets[15]. The LinkedOmics database was used to probe genes differentially expressed in correlation with TRIP13 in the TCGA BC cohort. presenting in volcano plots, heat maps. Correlation data results were signed , ranked, and used to GSEA perform analysis of Gene Ontology (GO). GO term can be divided into three parts: biological process (BP), cellular component (CC), and molecular function (MF). The rank criterion was the p-value < 0.01, FDR < 0.25, and 1000 simulations were performed.
Gene set enrichment analysis(GSEA)
Gene set enrichment analysis (GSEA) can be used to identify the biological mechanisms pathways according to the expression matrix in order. In order to probe the downstream signaling pathway correlate to TRIP13. The GSE2034 databset was divided into two groups based on the median expression level of TRIP13 expression. Then the GSEA software (v2.1.0, Broad Institute) would calculatedthe gene sets with a high enrichment score with TRIP13. Enrichment results were considered significant at with p value < 0.05 and FDR < 0.25.
PPI network construction analysis
String Database (https://string-db.org) is an online public database to gain insights into the functional associations between proteins[16, 17]. The protein-protein interaction (PPI ) network of TRIP13 was constructed by the String. Setting the a combined score > 0.7 were considered high confidence. Then, the PPI network was visualized by the Cytoscape software (version 3.5.1).
Patients tissue specimens
20 paraffin-embedded BC tissues and paired normal control were obtained from the Department of Pathology, Third Affiliated Hospital of Southern Medical University. All paraffin-embedded tissues were cut into 2.0 μm slices and transferred to glass slides for furder use. Informed consent of specimens was obtained from each patient before surgery. The study was approved by the ethics committee of The Third Affiliated Hospital of Southern Medical University.
Cell lines and animal models
Breast cancer cells 4T1 were obtained from the American Type Culture Collection (ATCC) and preserved in the Key Laboratory of Molecular Tumor Pathology, Southern Medical University. 4T1 was cultured in RPMI 1640 (GIBCO, USA) supplemented with 10% fetal bovine serum (HyClone, USA) at 37 °C in a humidified atmosphere of 5% CO2.
6-week-old female BALB/c mice were housed in a specific pathogen-free environment. All mice were purchased from the Animal Center of Southern Medical University , Guangzhou, China. 10 × 104 4T1 cells were injected into the mammary fat pad of each mouse. After 30 days, all mice were sacrificed and their primary tumor and lung were removed. Collected organs were fixed in formalin solution and then embedded in paraffin for further research. All study protocol for mice were approved by the Institutional Animal Care and Use Committee of Southern Medical University.
Immunohistochemical analysis
Specimens were embedded in paraffin, and then cut into 2.0 μm slices for immunohistochemical analysis. IHC staining was performed in our tissue sections by the by following protocol. First, the sections were stepwise dewaxed and ethanol to water. To block endogenous peroxidase activity, sections were immersed in the hydrogen peroxide solution. Subsequently, the sections were incubated with anti-TRIP13 antibodies (Proteintech, 19602-1-AP, 1:100 dilution) overnight at 4 °C. Next day, incubating secondary antibody 1 hour and then DAB solution 1 min at room temperature, sections were counterstained with hematoxylin for 5 min and washed by PBS three times for 5 min. Stained tissue sections were evaluated under a light microscope. The results were recorded as a sum of the staining intensity and percentage of positive tumor cells.
Statistical analysis
All statistical analyses were performed by default of the web resources. A two-tailed Student’s t-test and χ2 test were conducted to compare differences between the conditions using SPSS 22.0. Survival curves of TRIP13 expression in BC patients were analyzed using the Kaplan–Meier method. p-value < 0.05 was considered statistically significant. (*, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001).