Additional file 1 of ciRS-7 is a prognostic biomarker and potential gene therapy target for renal cell carcinoma

Additional file 1: Table S1. Full sequence information of ciRS-7; Table S2. PCR primer, siRNA and probe sequence; Table S3. Antibodies list; Table S4. The relationship between the expression of ciRS-7 and various clinicopathological variables; Table S5. Univariate and multivariate Cox regression analysis and the relationship between ciRS-7 expression and overall survival; Table S6. Univariate and multivariate Cox regression analysis and the relationship between miR-139-3p expression and overall survival; Figure S1. ciRS-7 was overexpressed in RCC tissues. A. Volcano plots analysis of differentially expressed circRNAs in GSE100186, GSE108735 and GSE137836. B. Agarose gel electrophoresis of PCR products of ciRS-7 in RCC cell lines. C. Relative expression of linear CDR1 was confirmed by qPCR in 786-O and ACHN cell lines transfected with control and siciRS-7#2. D. Detection of ciRS-7 expression after transfection with the indicated vectors by FISH. Nuclei were stained blue (DAPI), ciRS-7 was stained red. Figure S2. ciRS-7 acts as a sponge of miR-139-3p in RCC cells. A. Two potential miRNAs absorbed by ciRS-7 were predicted through circBank, miRanda, circAtlas and RNAhybrid. B. Relative expression of two miRNAs enriched by ciRS-7 probe lysates was detected by qRT-PCR. C. Relative expression of two miRNAs in TCGA RCC database. D. Three possible binding sites of miR-139-3p to ciRS-7. E. Dual luciferase reporter assay demonstrated that miR-139-3p is a direct target of ciRS-7. F and G. RNA pull-down assay shown that miR-139-3p is a direct target of ciRS-7. H. Detection of colocalization of ciRS-7 and miR-139-3p in cytoplasm by RNA FISH assay. Nuclei were stained blue (DAPI), ciRS-7 was stained red, and miR-139-3p was stained green, I. Correlations between ciRS-7 and miR-139-3p expression were found with Pearson’s correlation analysis in RCC tissue samples (n = 85). (**p < 0.01).  Figure S3. miR-139-3p was downregulated in TCGA KIRC database. A. Expression of miR-139-3p in normal and tumors tissues of TCGA RCC dataset. B. Expression of miR-139-3p in normal and paired tumors tissues of TCGA RCC dataset. C-H. Relative expression levels of miR-139-3p in TCGA RCC subgroup: gender (C) tumor stage (D) lymphatic invasion (E) metastasis status (F) tumor grade (G) and tumor stage (H). I. Overall survival curve of RCC patients with low and high miR-139-3p expression. J and K. Univariate and multivariate cox regression analyses of miR-139-3p expression with overall survival in TCGA database. Figure S4. miR-139-3p inhibits RCC cell proliferation, migration and invasion in vitro. A. miR-139-3p had low expression in RCC tumour tissues compared with adjacent normal tissues. B. Relative expression of miR-139-3p was confirmed by qPCR in 786-O and ACHN cell lines transfected with miR-139-3p-NC, miR-139-3p-Mimic or pcDNA3.1/miR-139-3p. C. Growth curves of 786-O and ACHN cell lines were measured by CCK-8. D. Wound healing assay to detect cell migration ability. E. Transwell assay to detect cell migration and invasion ability. F. Colony formation assay to detect cell migration ability. G. Edu assay to detect cell proliferation capacity. (**p < 0.01, ***p < 0.001). Figure S5. TAGLN is a target gene of ciRS-7, and ciRS-7 activates the PI3K/AKT signaling pathway. A. Heatmap of RNA-Seq analysis of sh-NC and sh-ciRS-7 cells. Red in the heatmap denotes upregulation, blue denotes downregulation. B. Venn diagram showing the number of genes that changes at the transcriptional or protein levels. C. colloidal Coomassie detects changes in protein levels in 786-O and ACHN cells. D. Dual luciferase reporter assay demonstrated that TAGLN is a direct target of miR-139-3p. E-G. Enrichment chord plot (E), GO bubble plot (F) and KEGG enrichment histogram (G) of down-regulated proteins. (**p <0.01). Figure S6. Sequencing of sh-NC and sh-ciRS-7 cells. A. Heatmap of lab-free quantitative of sh-NC and sh-ciRS-7 cells. Red in the heatmap denotes upregulation, green denotes downregulation. B and C. GO bubble plot (B) and KEGG enrichment histogram (C) of up-regulated proteins. Figure S7. ciRS-7 regulating the miR-139-3p/TAGLN axis and activating the PI3K/AKT signaling pathway to promote RCC cell proliferation, migration and invasion. A. The expression of TAGLN, p-PI3K and p-AKT were detected by western blot after overexpression or knockdown of ciRS-7. B. The expression of TAGLN, p-PI3K and p-AKT were detected by western blot after overexpression or knockdown of miR-139-3p. C. Rescue assay of miR-139-3p after overexpression of ciRS-7 in 786-O cells. D. Rescue assay of miR-139-3p after knockdown of ciRS-7 in 786-O cells. E. Growth curves of 786-O and ACHN cell lines were measured by CCK-8. F. Colony formation assay to detect cell migration ability. G. Edu assay to detect cell proliferation capacity. H. Transwell assay to detect cell migration and invasion ability. I. Wound healing assay to detect cell migration ability. (*p < 0.05, **p < 0.01, ***p < 0.001). Figure S8. Characteristics of PBAE. A. The 1HNMR analysis of PBAE. B, C. Growth curves of 786-O and ACHN cell lines were measured by CCK-8.