The ONCOMINE Database (www.oncomine.org) is an integrated online cancer microarray database that contains data from DNA sequencing and RNA sequencing (DNA-seq and RNA-seq, respectively) analysis, which are used to classify differential expressions between common cancer types and cancerous types. Corresponding normal tissue and clinical and pathological data. (18). In our study, the transcriptional expression data for E2F2 in different cancer tissue samples and their corresponding adjacent normal samples were obtained from the ONCOMINE database. Differences in transcriptional expression were compared by Student’s t test. The p-value cutoff and fold change threshold were as follows: p-value, 0.01; fold change, 1.5; gene grade, 10%; and data type, mRNA.
UALCAN (http://ualcan.path.uab.edu) is an interactive network resource developed based on level 3 RNA sequences and clinical data for 31 cancer types in the TCGA database (19). In this study, UALCAN was used to analyze the mRNA expression of E2F2 in primary gastric cancer tissue samples and the relationship between these members and clinicopathological parameters. The differences of transcriptional expression were compared by students't-test (p < 0.01).
The (GEPIA) database for interactive analysis of gene expression profiles (http://gepia.cancer-pku.cn/) is an interactive Web, containing 9736 tumors and 8587 normal samples from the TCGA and GTEx projects (20). In this study, GEPIA was used to verify the expression of E2F2 in GC.
The LinkedOmics database (http://www.linkedomics.org/login.php) is a Web-based platform for analyzing 32 cubes related to TCGA cancer (21). The Pearson co-expression was statistically analyzed by using E2F2 correlation coefficient, and displayed in the form of volcano map, heat map or scatter diagram. The rank criterion was FDR < 0.05 and 1000 simulations were performed.
cBioPortal includes 478 gastric cancer pathology reports data from TCGA (22). The genome map includes mutations, copy number changes from GISTIC (CNA), mRNA expression z score (RNA-seq V2 RSEM) and protein expression z score (RPPA). The E2F2 mutation, copy number variation (CNV) and gene co-occurrence in GC were analyzed by c-BioPortal tool.
TIMER (https://cistrome.shinyapps.io/timer/) is a comprehensive resource for systematic analysis of immune infiltration in various cancer types, including 10897 samples from 32 cancer types (23). TIMER used deconvolution method (24) to infer the abundance of tumor infiltrating immune cells (TIIC) from the gene expression profile. We analyzed the expression of E2F2 in STAD and the correlation between the expression of E2F2 and the content of immune infiltrating fluid.
7.Protein-protein interaction (PPI) network construction and gene enrichment analyses
In this study, the STRING database (25) was used to analyze E2F2 and 50 frequently changing genes closely related to family members. We used the annotated, visualization and comprehensive discovery database (DAVID) (http://www.DAVID.org) (26) to conduct an (KEGG) analysis of the motivational gene ontology (GO) and the Kyoto Encyclopedia of genes and genomes. GO enrichment analysis can predict gene function according to the composition of (CC) and molecular function (MF) of (BP), cells in biological process, while KEGG can be used to analyze the pathway of gene enrichment.
8.Tissue microarrays (TMAs)
We collected 60 fresh GC tissues and adjacent tissues for IHC staining and evaluation. The staining intensity was given a score from 0 to 3 based on the following criteria: 0, no staining; 1, weak staining; 2, moderate staining; or 3, strong staining. All individual patients understood the objectives of this study and provided written informed consent. The Medical Ethics Committee of Qingdao University and the Affiliated Hospital of Qingdao University approved the collection of clinical materials for research purposes.
9.Cells and culture conditions
AGS and HGC27 cell lines were purchased from the cell bank of the Chinese Academy of Sciences and were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (Gibco, NY, USA). The cells were placed in an incubator at 37 °C with a CO2 concentration of 5%.
Cells were transfected with plasmids expressing E2F2 (GV141-E2F2, Genechem, Shanghai, China), plasmids expressing empty vector (GV141-Vector), small interfering RNAs (siRNAs) against E2F2 (siE2F2; GenePharma, Shanghai, China, Supplementary Table 1) and negative control (siNC) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
11.RNA extraction and qPCR
RNA was extracted from cells via Trizol (TaKaRa). 1μg of this isolated RNA was then used to generate cDNA using 5 × primer script buffer, primer script enzymes, oligo dT primers and reverse transcriptase (TaKaRa). cDNA was generated through a reverse transcription reaction which was conducted at 37 °C for 15 min, 84 °C for 5 s and 4 °C for the appropriate amount of time. Real-time quantitative PCR (qPCR) was carried out using SYBR Green PCR master mix. Gene expression was normalized against β-actin. Each experiment was repeated three times and was performed independently in duplicate. mRNA expression was quantified by the cycle threshold (CT) method, and SPSS version 11.5 was used to calculate identify significant differences in the mRNA expression levels of various genes among different samples using the Mann-Whitney U test. The PCR primers used are listed in Supplementary Table 1.
12.Western blotting analysis
Western blotting analysis of protein expression was performed as described previously (27). Briefly, protein lysates (20 µg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and target proteins were detected by western blotting with antibodies against E2F2 (1:1000) and GAPDH (1:50,000). Other antibodies used in this study are listed in Supplementary Table 1.
The cells were fixed at 4 ℃ with 2.5% glutaraldehyde (Solarbio, Beijing, China). H, washed with phosphate buffer (PBS) and fixed with 1%OsO4 buffer at 4 ℃. H . The cells were then washed and dehydrated in a series of graded ethanol solutions and embedded in Epon812 epoxy resin. Ultra-thin (90 nm) sections were collected on copper net, stained with 1% uranyl acetate and 0.2% lead citrate, and examined by JEOL-1200EX transmission electron microscope (Beijing, China).
14.Cell migration and invasion assays
Cells in logarithmic phase were starved in serum-free medium 1640 for 24 hours, then digested with 0.25% EDTA-trypsin. The cell suspension was then treated with serum-free 1640, and 200 μ L of cells were added to the upper chamber of the Transwell insert (Corning Costar), while the complete growth medium containing serum was added to the lower chamber at a rate of 600 μ L / L. After 24 hours, the cells were fixed with methanol for 20 minutes, and then stained with crystal violet for 20 minutes after drying. Use a wet swab to gently remove the cells retained in the upper chamber, and then place the chamber under an inverted microscope so that the remaining cells can be counted. The images were analyzed by ImageJ software.
We inoculated the cells into a 6-well plate and then used a 200 μ L pipette suction head to form a wound on the monolayer surface of the fused cells. The cells were then washed with PBS and incubated in serum-free medium for 24 hours. Since then, we have captured images of the cells at different times within 24 hours. The width of the wound at × 100 magnification was evaluated by microscope. The length of the wound was measured at random intervals and the data were analyzed by ImageJ software.
Data are means ± standard deviation. Two-tailed unpaired Student’s t tests were used to assess significance unless stated otherwise. P < 0.05 was deemed significant.