Additional file 2 of Circular RNA circFIRRE drives osteosarcoma progression and metastasis through tumorigenic-angiogenic coupling

Additional file 2: Figure S1. Validation of differentially expressed circRNAs in RNA-seq. (A) The length distribution of aberrantly expressed circRNAs in RNA-seq. (B) 30 upregulated circRNAs were screened out through filter criteria as described and sorted by name. (C) Quantification of the fluorescence intensity of circFIRRE in FISH assay (n=5 in each group). (D) Online prediction algorithms (lncLocator, www.csbio.sjtu.edu.cn/bioinf/lncLocator) was applied to explore the intracellular localization of circFIRRE in OS cells. (E) Quantification of the fluorescence intensity of circFIRRE in both nucleus and cytoplasm in FISH assay (n=15 in each group). Figure S2. Baseline clinical data and GSEA analysis data. (A) Clinical baseline characteristics of 104 OS patients. (B-F) GSEA (https://www.gsea-msigdb.org/gsea/index.jsp) analysis of hallmark gene sets showed that highly expressed circFIRRE was associated with epithelial mesenchymal transition (EMT), cell cycle (E2F targets, G2M checkpoint and mitotic spindle) and angiogenesis.  Figure S3. circFIRRE knockdown can inhibit OS progression in vitro. (A) Three circFIRRE-specific shRNAs were designed and the knockdown efficiency were verified by RT-qPCR in both MG63 and U2OS (n=3 in each group). (B) CCK8 assay was applied to estimate cell viability influenced after transient transfection of siRNAs (si-circFIRRE-1 and -2) at different time points in both MG63 and U2OS (n=6 at each time point). (C) Wound healing assay exhibited cell migration. Scar bar=200 μm. (D) Schematic of the lentiviral vector GV344 (hU6-MCS-Ubiquitin-firefly_Luciferase-IRES-puromycin). (E) Wound healing assay exhibited cell migration after stable infection of lentivirus in MG63 and U2OS cells. Scar bar=200 μm. (F-H) Flow cytometry analysis of cell cycle distribution. (G-H) Quantification of cell cycle in MG63 and U2OS cells (n=3 in each group). Values are presented as mean ± SD; the bar charts, line charts, error bars and dots represent the quantitative analysis of 3 independent experiments; two-way ANOVA were used; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns = not significant. Figure S4. circFIRRE overexpression can promote OS progression in vitro. (A) Schematic of the circFIRRE overexpression vector GM-7183. (B) Sanger sequencing was applied to verify back-spliced junction of circFIRRE (GGAG) after vector construction. (C) Wound healing assay exhibiting cell migration. Scar bar=200 μm. (D-F) Flow cytometry assays presenting cell cycle distribution. Values are presented as mean ± SD; the bar charts, error bars and dots represent the quantitative analysis of 3 independent experiments; two-way ANOVA were used; *P < 0.05; **P < 0.01. Figure S5. circFIRRE knockdown can inhibit angiogenesis. (A) CCK8 assay was applied to estimate cell viability influenced after transient transfection of siRNAs (si-circFIRRE-1 and -2) at different time points in HUVEC cells (n=6 at each time point). (B-C) EdU assay was performed to estimate cell proliferation after circFIRRE silencing in HUVEC cells. S-phase entry is visualized by EdU incorporation (green); DAPI-stained nuclei (blue). Scar bar=200 μm. (C) Quantification was conducted as described (n=5 in each group). (D-F) Wound healing and Tranwell migration assays exhibited cell migration after circFIRRE silencing in HUVEC cells. Scar bars=200 μm and 400 μm. (F) Quantification of Transwell assay was conducted as described (n=5 in each group). (G) White light micrographs (top) and highlighted microvessel areas (red) in fluorescence micrographs (bottom) at the same fields of Figure 4G. Scar bars=1 mm and 400 μm. (H) Quantification of aortic ring microvessel area compared to negative control aorta (n=5 in each group). (I) White light images of CAM photographed in fertilized eggs at the same fields of Figure 4H. (J) The statistical results of the CAM assay (n=5 in each group). Values are presented as mean ± SD; the bar charts, line charts, error bars and dots represent the quantitative analysis of 3 independent experiments; (C, F, H, one-way ANOVA; J, two-way ANOVA); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Figure S6. circFIRRE overexpression can promote angiogenesis. (A) Relative circFIRRE expression was detected by RT-qPCR after overexpression vectors or scramble vectors transfection in HUVEC cells (n=3 in each group). (B-D) CCK8 and EdU assays exhibited cell proliferation in HUVEC under circFIRRE overexpression. Scar bar=200 μm. (E-G) Wound healing and Transwell migration assays exhibited cell migration in HUVEC under circFIRRE overexpression. Scar bars=200 μm and 400 μm. (H) White light micrographs (top) and highlighted microvessel areas (red) in fluorescence micrographs (bottom) at the same fields of Figure 4K. Scar bars=1mm and 400 μm. (I) Quantification of aortic ring microvessel area compared to negative control aorta (n=5 in each group). (J) White light images of CAM photographed in fertilized eggs at the same fields of Figure 4L. (K) The statistical results of the CAM assay (n=5 in each group). Values are presented as mean ± SD; the bar charts, line charts, error bars and dots represent the quantitative analysis of 3 independent experiments; (A, D, G, I, 2-tailed Student t test; B, K, two-way ANOVA); *P < 0.05; **P < 0.01; ****P < 0.0001. Figure S7. circFIRRE is induced by YY1. (A-B) Relative expression of YY1 and gene FIRRE was upregulated in sarcoma (SARC) in TCGA database. (C-E) The FPKM of YY1, gene FIRRE and linear FIRRE in RNA-seq. (F) Fold change of linear FIRRE and circFIRRE in RNA-seq.  Figure S8. circFIRRE can sponge miR-486-3p and miR-1225-5p. (A-B) Relative expression of miR-486-3p and miR-1225-5p was examined by RT-qPCR in 5 OS cell lines and normal osteoblasts (n=3 in each group). (C-D) Preliminary experiments to test circFIRRE probe by RT-qPCR and RT-PCR in both MG63 and U2OS cells (n=3 in each group). (E) The predicted binding sites of miR-486-3p and miR-1225-5p in circFIRRE. (F) Validation of FIRRE siRNAs knockdown in MG63 and U2OS (n=3 in each group). (G) Relative expression of miR-486-3p and miR-1225-5p was examined by RT-qPCR under FIRRE knockdown (n=3 in each group). (H-I) Preliminary experiments to test FIRRE probe by RT-qPCR and RT-PCR in both MG63 and U2OS cells (n=3 in each group). (J) Relative expression of miR-486-3p and miR-1225-5p was examined by RT-qPCR under FIRRE pull-down (n=3 in each group). Values are presented as mean ± SD; the bar charts, error bars and dots represent the quantitative analysis of 3 independent experiments in A, B; (A, B, one-way ANOVA; C, F, G, H, J, two-way ANOVA); **P < 0.01; ***P < 0.001; ****P < 0.0001. Figure S9. LUZP1 knockdown can inhibit tumor proliferation, progression and angiogenesis. (A) The FPKM of LUZP1 in RNA-seq. (B) Relative expression of LUZP1 in sarcoma (SARC) in TCGA database. (C-D) Validation of LUZP1 siRNA knockdown in MG63 and U2OS. (E-F) CCK8 assay was applied to estimate cell viability influenced by LUZP1 knockdown at different time points in both MG63 and U2OS (n=6 at each time point). (G-J) Transwell migration and invasion assays were employed to detect cell migration and invasion abilities influenced by LUZP1 knockdown (n=3 in each group). Scar bar=400 μm. (K-L) Tube formation assay was applied to determine cell tube formation ability influenced by LUZP1 knockdown in HUVEC cells. Scar bar=1mm. Values are presented as mean ± SD; the bar charts, line charts, error bars and dots represent the quantitative analysis of 3 independent experiments; two-way ANOVA were used; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Figure S10. circFIRRE promotes OS tumorigenesis by miR-486-3p/miR-1225-5p-LUZP1 axis in vitro. (A) The predicted binding sites of miR-486-3p and miR-1225-5p in LUZP1. (B-E) The mRNA and protein level of LUZP1 after circFIRRE silencing or overexpression. (F) CCK8 assay exhibited cell proliferation in MG63 and U2OS cells (n=3 in each group). (G) Wound healing assay exhibited cell migration in MG63 and U2OS cells. Scar bar=200 μm. Values are presented as mean ± SD; the bar charts, line charts, error bars and dots represent the quantitative analysis of 3 independent experiments; two-way ANOVA were used; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Figure S11. Representative IHC staining of primary OS lesions in xenograft models (E-cadherin, N-cadherin and Vimentin). Scar bars=100 μm and 50 μm. Figure S12. Representative FISH images of lung metastatic lesions exhibited circFIRRE, miR-486-3p and miR-1225-5p expression in xenograft models. Scar bar=100 μm. Figure S13. Representative IHC staining of lung metastatic lesions in xenograft models (ki-67, E-cadherin, N-cadherin and Vimentin). Scar bars=100 μm and 50 μm.