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.

附加文件2：图S1。RNA测序中差异表达环状RNA（circRNA）的验证。(A) RNA测序中异常表达环状RNA的长度分布。(B) 按照既定筛选标准筛选出30条上调环状RNA，并按名称排序。(C) 荧光原位杂交（FISH）实验中circFIRRE的荧光强度定量分析（每组n=5）。(D) 使用在线预测算法（lncLocator，www.csbio.sjtu.edu.cn/bioinf/lncLocator）探究circFIRRE在骨肉瘤（OS）细胞中的细胞内定位。(E) 荧光原位杂交实验中细胞核与细胞质内circFIRRE的荧光强度定量分析（每组n=15）。图S2。基线临床数据与基因集富集分析（GSEA）数据。(A) 104例骨肉瘤患者的临床基线特征。(B-F) hallmark基因集的基因集富集分析（https://www.gsea-msigdb.org/gsea/index.jsp）结果显示，高表达circFIRRE与上皮间质转化（EMT）、细胞周期（E2F靶点、G2M检查点及有丝分裂纺锤体）及血管生成相关。图S3。敲低circFIRRE可在体外抑制骨肉瘤进展。(A) 设计3条circFIRRE特异性短发夹RNA（shRNA），并通过实时定量聚合酶链反应（RT-qPCR）验证其在MG63和U2OS细胞中的敲低效率（每组n=3）。(B) 采用细胞计数试剂盒-8（CCK8）实验，检测不同时间点瞬时转染小干扰RNA（si-circFIRRE-1和-2）后MG63和U2OS细胞的细胞活力（每个时间点n=6）。(C) 划痕愈合实验显示细胞迁移能力。标尺=200 μm。(D) 慢病毒载体GV344（hU6-MCS-Ubiquitin-firefly_Luciferase-IRES-puromycin）示意图。(E) MG63和U2OS细胞经慢病毒稳定感染后，划痕愈合实验显示细胞迁移能力。标尺=200 μm。(F-H) 流式细胞术分析细胞周期分布。(G-H) MG63和U2OS细胞的细胞周期定量分析（每组n=3）。数据以平均值±标准差表示；柱状图、折线图、误差棒及散点代表3次独立实验的定量结果；采用双因素方差分析（two-way ANOVA）；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001；ns = 无显著性差异。图S4。过表达circFIRRE可在体外促进骨肉瘤进展。(A) circFIRRE过表达载体GM-7183示意图。(B) 载体构建后，采用桑格测序验证circFIRRE的反向剪接接头（GGAG）。(C) 划痕愈合实验显示细胞迁移能力。(D-F) 流式细胞术分析细胞周期分布。数据以平均值±标准差表示；柱状图、误差棒及散点代表3次独立实验的定量结果；采用双因素方差分析；*P < 0.05；**P < 0.01。图S5。敲低circFIRRE可抑制血管生成。(A) 采用CCK8实验检测不同时间点瞬时转染si-circFIRRE-1和-2后人脐静脉内皮细胞（HUVEC）的细胞活力（每个时间点n=6）。(B-C) EdU实验检测circFIRRE沉默后人脐静脉内皮细胞的增殖能力：EdU掺入标记S期细胞（绿色）；DAPI染色细胞核（蓝色）。标尺=200 μm。(C) 按照既定方法进行定量分析（每组n=5）。(D-F) 划痕愈合及Transwell迁移实验显示circFIRRE沉默后人脐静脉内皮细胞的迁移能力。标尺分别为200 μm和400 μm。(F) 按照既定方法进行Transwell实验的定量分析（每组n=5）。(G) 同图4G视野下的明场显微照片（上）及荧光显微照片中高亮的微血管区域（红色，下）。标尺分别为1 mm和400 μm。(H) 与阴性对照主动脉相比，主动脉环微血管面积的定量分析（每组n=5）。(I) 同图4H视野下的受精卵鸡胚尿囊膜（CAM）明场照片。(J) 鸡胚尿囊膜实验的统计结果（每组n=5）。数据以平均值±标准差表示；柱状图、折线图、误差棒及散点代表3次独立实验的定量结果；(C、F、H采用单因素方差分析；J采用双因素方差分析)；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。图S6。过表达circFIRRE可促进血管生成。(A) 采用RT-qPCR检测人脐静脉内皮细胞中转染过表达载体或空载体后circFIRRE的相对表达量（每组n=3）。(B-D) CCK8及EdU实验显示circFIRRE过表达后人脐静脉内皮细胞的增殖能力。标尺=200 μm。(E-G) 划痕愈合及Transwell迁移实验显示circFIRRE过表达后人脐静脉内皮细胞的迁移能力。标尺分别为200 μm和400 μm。(H) 同图4K视野下的明场显微照片（上）及荧光显微照片中高亮的微血管区域（红色，下）。标尺分别为1 mm和400 μm。(I) 与阴性对照主动脉相比，主动脉环微血管面积的定量分析（每组n=5）。(J) 同图4L视野下的受精卵鸡胚尿囊膜明场照片。(K) 鸡胚尿囊膜实验的统计结果（每组n=5）。数据以平均值±标准差表示；柱状图、折线图、误差棒及散点代表3次独立实验的定量结果；(A、D、G、I采用双侧Student t检验；B、K采用双因素方差分析)；*P < 0.05；**P < 0.01；****P < 0.0001。图S7。circFIRRE受YY1诱导。(A-B) 癌症基因组图谱（TCGA）数据库中肉瘤（SARC）组织内YY1及FIRRE基因的相对表达量均上调。(C-E) RNA测序中YY1、FIRRE基因及线性FIRRE的每千碱基百万片段数（FPKM）。(F) RNA测序中线性FIRRE及circFIRRE的倍数变化。图S8。circFIRRE可作为miR-486-3p和miR-1225-5p的分子海绵。(A-B) 采用RT-qPCR检测5株骨肉瘤细胞系及正常成骨细胞中miR-486-3p和miR-1225-5p的相对表达量（每组n=3）。(C-D) 在MG63和U2OS细胞中通过RT-qPCR及逆转录聚合酶链反应（RT-PCR）验证circFIRRE探针的初步实验（每组n=3）。(E) circFIRRE中miR-486-3p和miR-1225-5p的预测结合位点。(F) 验证FIRRE siRNA在MG63和U2OS细胞中的敲低效率（每组n=3）。(G) 采用RT-qPCR检测FIRRE敲低后miR-486-3p和miR-1225-5p的相对表达量（每组n=3）。(H-I) 在MG63和U2OS细胞中通过RT-qPCR及RT-PCR验证FIRRE探针的初步实验（每组n=3）。(J) 采用RT-qPCR检测FIRRE下拉实验后miR-486-3p和miR-1225-5p的相对表达量（每组n=3）。数据以平均值±标准差表示；A、B中的柱状图、误差棒及散点代表3次独立实验的定量结果；(A、B采用单因素方差分析；C、F、G、H、J采用双因素方差分析)；**P < 0.01；***P < 0.001；****P < 0.0001。图S9。敲低LUZP1可抑制肿瘤增殖、进展及血管生成。(A) RNA测序中LUZP1的每千碱基百万片段数（FPKM）。(B) TCGA数据库中肉瘤组织内LUZP1的相对表达量。(C-D) 验证LUZP1 siRNA在MG63和U2OS细胞中的敲低效率。(E-F) 采用CCK8实验检测不同时间点LUZP1敲低后MG63和U2OS细胞的细胞活力（每个时间点n=6）。(G-J) 采用Transwell迁移及侵袭实验检测LUZP1敲低后细胞的迁移及侵袭能力（每组n=3）。标尺=400 μm。(K-L) 采用管形成实验检测LUZP1敲低后人脐静脉内皮细胞的管形成能力。标尺=1 mm。数据以平均值±标准差表示；柱状图、折线图、误差棒及散点代表3次独立实验的定量结果；采用双因素方差分析；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。图S10。circFIRRE通过miR-486-3p/miR-1225-5p-LUZP1轴在体外促进骨肉瘤发生。(A) LUZP1中miR-486-3p和miR-1225-5p的预测结合位点。(B-E) 敲低或过表达circFIRRE后LUZP1的mRNA及蛋白水平。(F) CCK8实验显示MG63和U2OS细胞的增殖能力（每组n=3）。(G) 划痕愈合实验显示MG63和U2OS细胞的迁移能力。标尺=200 μm。数据以平均值±标准差表示；柱状图、折线图、误差棒及散点代表3次独立实验的定量结果；采用双因素方差分析；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。图S11。异种移植模型中原发性骨肉瘤病灶的代表性免疫组化（IHC）染色（E-钙粘蛋白、N-钙粘蛋白及波形蛋白）。标尺分别为100 μm和50 μm。图S12。异种移植模型中肺转移病灶的代表性荧光原位杂交图像，显示circFIRRE、miR-486-3p及miR-1225-5p的表达。标尺=100 μm。图S13。异种移植模型中肺转移病灶的代表性免疫组化染色（ki-67、E-钙粘蛋白、N-钙粘蛋白及波形蛋白）。标尺分别为100 μm和50 μm。