Fig 2: MiR-599 directly interacted with ONECUT2. (A) ONECUT2 was predicted to be the target gene of miR-599. (B-E) The combination between miR-599 and ONECUT2 was assessed by RIP and RNA pull down assays. (F) The binding sites between ONECUT2 and miR-599. (G and H) The combination between miR-599 and ONECUT2 was analyzed by dual-luciferase reporter assay. (I and G) The mRNA and protein levels of ONECUT2 in SW620 and DLD-1 cells transfected with miR-NC or miR-599 were measured by qRT-PCR assay or western blot assay. (K and L) The mRNA and protein levels of ONECUT2 in SW620 and DLD-1 cells transfected with sh-NC + anti-NC, sh-circ_0084615 + anti-NC or sh-circ_0084615 + anti-miR-599 were measured by qRT-PCR assay or western blot assay. (M and N) The expression of ONECUT2 in CRC tissues and normal tissues was examined with qRT-PCR and IHC. (O and P) The mRNA and protein levels of ONECUT2 in FHC, SW620, DLD1, SW480 and HT-29 cells were measured by qRT-PCR assay or western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 3: MiR-599 overexpression altered CRC cell growth, motility and angiogenesis by binding to ONECUT2. (A) The protein level of ONECUT2 in SW620 and DLD-1 cells transfected with ONECUT2 or vector was measured via western blot assay. (B-H) SW620 and DLD-1 cells were transfected with miR-NC + vector, miR-599 + vector or miR-599 + ONECUT2. (B-F) The proliferation, colony formation, migration, invasion and angiogenesis of SW620 and DLD-1 cells were investigated by EdU assay, colony formation assay, wound healing assay, transwell assay and tube formation assay, respectively. (G and H) The protein levels of ZEB2, E-cadherin, Vimentin and VEGFA in SW620 and DLD-1 cells were measured via western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 4: TMEscore was an independent factor for predicting patient survival. (A) The identification flowchart of six prognostic genes (C6orf223, EPHX4, HES6, NKD2 OLR1 and ONECUT2). (B) The expression of the six genes in GIAC tumor and normal samples from TCGA cohort. (C) The RNA expression of the six genes in 55 pairs of CRC tumor and normal samples from our center (GAD and ESAD see in Supplementary Figures 7, 8). (D) The expression of the five proteins in 10 pairs of CRC tumor and normal samples from our center (GAD and ESAD see in Supplementary Figures 9, 10). (E) The WB image of protein expression for CRC (GAD and ESAD see in Supplementary Figures 11, 12). (F–K) The survival time (OS and PFS) of patients with high/low TMEscore in TCGA cohort (F, G), GSE17536 (H, I) and GSE39582 (J, K).