Integrative analyses of bulk microarray data to discover genes, pathways, and immune infiltration characteristics associated with targeting of Ewing sarcoma

Fig. 1. Study workflow shows overall steps carried out in the integrated microarray analyses

Fig. 2. Identification of differentially expressed genes (DEGs) and enriched pathways in ES. (A) A volcano plot of DEGs in tumor samples compared to normal samples. Red indicates the gene expression was up-regulated in tumor samples compared to normal samples (P < 0.05 and logFC > 1); green indicates the gene expression was down-regulated in tumor samples compared with normal samples (P < 0.05 and logFC < -1); Black indicates the |logFC|< 1. (B–D) GO analysis. The size of the dots represents the gene ratio. E KEGG pathway enrichment analysis. The size of the dots represents the number of DEGs in the pathway. F GSEA analysis. Red indicates NES > 0; green indicates NES < 0. The size of the dots represents the number of DEGs in the pathway. (G–H) The GSEA plot of the two pathways: G2M checkpoint and E2F Targets

Fig. 3. Analysis of association between DEGs and survival. (A) Screening of DEGs. A total of 138 overlapping DEGs were identified in the two datasets which satisfied the criteria HR > 1 and logFC > 1. A total of 38 overlapping DEGs were identified in the two datasets which satisfied the criteria HR < 1 and logFC < -1. B Heatmap of the top ten overlapping DEGs (10 + 10) mentioned above. C The samples were grouped based on the expression level of the E2F-Targets geneset, and survival analysis was performed using GSE17618 survival information. The red and blue lines represent two different groups. D Gene expression heatmap of E2F-Targets geneset for grouped samples

Fig. 4. Immune infiltration analysis results. Significant highly (A) and lowly (B) immune infiltrated lymphocytes in tumor than those in normal samples are shown

Fig. 5. In vitro verification of four up-regulated DEGs with decreased survival. (A) Comparison of expression of four DEGs (ASPM, CENPF, CKS2, and DTL) between tumor and normal samples. Normal samples (N): blue, tumor samples (T): red. B The Kaplan–Meier survival curves of the selected four genes. C RT‑qPCR verification of silencing efficiencis of siRNAs targeting the respective four genes. (D) Cell proliferation assay tesing knock-down effects of the four genes. A-673 cells were cultured in six-well plates and transfected with siRNAs, respectively. Cell counts were performed on day 1, day 2 and day 3. Data are shown as mean ± SD, n = 3 independent experiments. (E–F) Clonogenic assay testing knock-down effects of the four genes. 500 cells with transfection of respective siRNAs were transferred to six-well plates and the cell culture was maintained for 14 days for colony formation observation. E Representative images of cell colonies. F Quantitative results of colony-forming assay. Data (Fig. 5A) are shown as mean ± SD, n = 22 (normal) v.s. n = 479 (tumor). Data (Fig. 5C, D, F) are shown as mean ± SD, n = 3 independent experiments. (ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001)