Fig 1: CCAT2 upregulates the expression of IGF2BP2 by adsorbing miR-200b to promote the migration and invasion of ESCC cells. (A) Venn diagram of differentially expressed genes from GEPIA analysis and downstream genes of miR-200b obtained by miRWalk, and the intersection genes are CXCL8, LAMC2, IGF2BP2, and EPHB2. (B) The binding site between miR-200b and IGF2BP2 predicted by the RNA22 database. (C) The box diagram of IGF2BP2 expression through GEPIA analysis; the left red box indicates the expression in ESCC samples, and the right gray box indicates the expression in normal samples. (D) RT-qPCR detection of mRNA expression of IGF2BP2 in 93 cases of ESCC tissues and normal adjacent tissues. (E) Correlation analysis of IGF2BP2 mRNA expression and miR-200b expression in 93 cases of ESCC tissues by Pearson’s correlation analysis. (F) Immunoblotting analysis of IGF2BP2 protein in 93 cases of ESCC tissues and normal adjacent tissues. (G) Binding of miR-200b to IGF2BP2 confirmed by dual luciferase report assay. (H, I) RT-qPCR and immunoblotting detection of the mRNA and protein expression of IGF2BP2 in ESC410 cells after overexpression of CCAT2 and miR-200b. (J) Scratch assay detection of ESC410 cell migration ability in each group. (K) Transwell assay detection of ESC410 cell invasion ability in each group, scale bar = 50 μm. *p < 0.05 vs. normal adjacent tissues, HET-1A cell line, oe-NC-treated cells, sh-NC-treated cells, or oe-NC + mimic NC-treated cells. # p < 0.05 vs. oe-CCAT2 + mimic NC-treated cells or oe-NC + miR-200b mimic-treated cells. Data were shown as mean ± standard deviation of three technical replicates. Data between cancer tissues and normal adjacent tissues were compared by paired t-test. Data between remaining two groups were compared by unpaired t-test. Data among multiple groups were compared by one-way ANOVA with Tukey’s post hoc test.
Fig 2: CCAT2 upregulates the expression of TK1 to promote the tumorigenesis of ESCC cells in nude mice. (A) Representative images showing xenografts in nude mice of each group. (B) Changes in the volume of formed tumors within four weeks of implanting ESC410 cells transfected with different plasmids in each group. (C) The weight of formed tumors within four weeks of implanting ESC410 cells in each group. (D, E) RT-qPCR detection of the expression of CCAT2 and miR-200b in cancer tissues of oe-NC + sh-NC-treated mice, oe-CCAT2 + sh-NC-treated mice, and oe-CCAT2 + sh-TK1-treated mice. (F, G) RT-qPCR and immunoblotting detection of mRNA and protein expressions of IGF2BP2 and TK1 in cancer tissues of each group. (H) Me-RIP detection of the m6A modification level of TK1 in cancer tissues of each group. *p < 0.05 vs. oe-NC + sh-NC-treated mice. # p < 0.05 vs. oe-CCAT2 + sh-NC-treated mice. Data were shown as mean ± standard deviation of three technical replicates. Data among multiple groups were compared by one-way ANOVA with Tukey’s post hoc test. Comparison among groups at different time points was performed using repeated measures ANOVA with Bonferroni’s post hoc test. ns means no significant difference.
Fig 3: IGF2BP2 maintains TK1 mRNA stability to promote TK1 expression by recognizing the m6A modification of TK1 mRNA. (A) Venn diagram of differentially expressed genes and related genes of IGF2BP2 obtained from Ualcan, LinkedOmics, GEPIA, and MEM, and the six intersection genes are FSCN1, ECT2, TPX2, MCM2, FANCI, and TK1. (B) Correlation map of IGF2BP2 expression and TK1 expression obtained by GEPIA analysis (p = 1.6e-07). (C) The box diagram of TK1 expression through GEPIA analysis; the left red box indicates the expression in ESCC samples, and the right gray box indicates the expression in normal samples. (D) RT-qPCR detection of mRNA expression of TK1 in 93 cases of ESCC tissues and normal adjacent tissues. (E) Correlation analysis of TK1 mRNA expression and IGF2BP2 mRNA expression in 93 cases of ESCC tissues by Pearson’s correlation analysis. (F, G). Immunoblotting detection of TK1 protein in 93 cases of ESCC tissues and normal adjacent tissues. (H) Me-RIP detection of the m6A modification level of TK1 in ESCC tissues and normal adjacent tissues. (I) PAR-CLIP detection of the binding between IGF2BP2 and TK1 mRNA in ESCC tissues and normal adjacent tissues. (J, K). RT-qPCR and immunoblotting detection of the mRNA and protein expressions of TK1 in oe-NC-treated and oe-IGF2BP2-treated cells. (L) Me-RIP detection of the m6A modification level of TK1 in oe-NC-treated and oe-IGF2BP2-treated cells. (M) PAR-CLIP detection of the binding between IGF2BP2 and TK1 mRNA in ESC410 cells of each group. (N, O) RT-qPCR and immunoblotting detection of the mRNA and protein expressions of TK1 in sh-NC-treated and sh-IGF2BP2-treated cells. (P) Me-RIP detection of the m6A modification level of TK1 in sh-NC-treated and sh-IGF2BP2-treated cells. (Q) PAR-CLIP detection of the binding between IGF2BP2 and TK1 mRNA in sh-NC-treated and sh-IGF2BP2-treated cells. *p < 0.05 vs. normal adjacent tissues, oe-NC-treated cells, or sh-NC-treated cells. Data were shown as mean ± standard deviation of three technical replicates. Data between cancer tissues and normal adjacent tissues were compared by paired t-test. Data between the remaining two groups were compared by unpaired t-test.
Fig 4: CCAT2 upregulates TK1 to promote the migration and invasion of ESCC cells in vitro. (A, B). RT-qPCR detection of the expression of CCAT2 and miR-200b in oe-NC + sh-NC-, oe-CCAT2 + sh-NC-, and oe-CCAT2 + sh-TK1-treated ESC410 cells. (C, D). RT-qPCR and immunoblotting detection of the mRNA and protein expression of IGF2BP2 and TK1 in ESC410 cells of each group. (E) Me-RIP detection of m6A modification level of TK1 in ESC410 cells of each group. (F) Scratch assay detection of the migration ability of ESC410 cells in each group. (G) Transwell assay detection of the invasion ability of ESC410 cells in each group, scale bar = 50 μm. *p < 0.05 vs. oe-NC + sh-NC-treated cells; # p < 0.05 vs. oe-CCAT2 + sh-NC-treated cells; ns indicates no significant difference. Data were shown as mean ± standard deviation of three technical replicates. Data between two groups were compared by unpaired t-test. Data among multiple groups were compared by one-way ANOVA with Tukey’s post hoc test.
Fig 5: Schematic diagram of the mechanism by which CCAT2 affects development and progression of ESCC. CCAT2 binds with miR-200b to reduce its expression, and consequently enhances the expression of the miR-200b target IGF2BP2, which leads to upregulated TK1 expression and promotion of migration and invasion of ESCC cells in vitro.
Supplier Page from Abcam for Recombinant Human IGF2BP2/IMP-2 protein