Fig 2: PIM1 knockdown promotes the nuclear relocation of endogenous RUNX3 and its transcriptional activity. A and C, Immunofluorescence images showing the localization of endogenous RUNX3 in MCF‐7, T47D, BT‐549 and MDA‐MB‐231 cells transfected with NC or siPIM1; B, Indicated the percentage of nuclear/cytoplasmic expression of RUNX3 in A, (D) indicated that of RUNX3 in C. E, Western blot analysis showing PIM1 knockdown promoted nuclear expression of endogenous RUNX3 in MCF‐7, T47D, MDA‐MB‐231 and BT‐549 cells. F, Real‐time qPCR analysis showing that PIM1 knockdown promoted the transcription of RUNX3 target gene Cdkn1a (p21) (n = 3). G, Luciferase assay showing the transcriptional activity of Cdkn1a (p21) promoter was improved when knocking down PIM1 (n = 3). H, Western blot analysis showing p21 expression was rescued after overexpressing RUNX(4A). Data were expressed as means ± SE of three independent experiments. Statistical significance was assessed using unpaired Student's t test and one‐way ANOVA (***P < .001; **P < .01; and *P < .05)

Fig 3: Upregulation of RUNX3 decreases the invasion and migration abilities of ESCC cells and decreases MMP-9 expression. (A) Representative images of cell migration (original magnification ×200). (B) Representative percentages of cell migration. (C) Representative images of cell invasion (original magnification ×200). (D) Representative percentages of cell invasion. Vector, vector group; RUNX3, RUNX3 group. *P<0.05, **P<0.01 and ***P<0.001. Upregulation of RUNX3 decreases the invasion and migration abilities of ESCC cells and decreases MMP-9 expression. (E) Representative images of the wound-healing assay performed with Eca109 and EC9706 cells; the scale bars represent 100 µm. (F and G) Effect of RUNX3 on MMP-9 expression in Eca109 and EC9706 cells. Vector, vector group; RUNX3, RUNX3 group. *P<0.05, **P<0.01 and ***P<0.001. RUNX3, runt-related transcription factor 3; ESCC, esophageal squamous cell carcinoma; matrix metallopeptidase 9.

Fig 4: A comparison of expression levels of RUNX genes in human cancers of different types. (A–C) RUNX1, RUNX2 and RUNX3 gene levels in different cancer types (red) and normal tissue (blue) available in TIMER database. (D–F) RUNX1, RUNX2 and RUNX3 gene levels in different cancer types (red) and normal tissue (blue) available in UALCAN database. (G–I) RUNX1, RUNX2 and RUNX3 gene levels in different cancer types (red) and normal tissue (blue) available in GEPIA database. (J) Pan-cancer landscape of differential expression of RUNX1, RUNX2, and RUNX3 across three different TCGA databases. *P< 0.05, **P< 0.01, and ***P< 0.001.

Fig 5: RUNX3 increases tumor formation in KGN cells. KGN/Vector and KGN/RUNX3 cells (2 × 107 cells) were injected subcutaneously into the left and right flank, respectively, of female NSG (NOD-scid IL2R-gammanull) mice twice over an interval of two weeks (n = 6). (A) Image of one KGN/RUNX3 tumor is shown. The left panel shows the location of the tumor. The right panel shows the image of the same tumor at a higher magnification. The scale bar is 4 mm. (B) Four KGN/RUNX3 tumors were harvested, dissociated into single cells, and passaged in culture. RUNX3 expression in the tumor-derived cells was examined by immunoblotting using RUNX3 and FLAG antibodies. β-actin was used as the loading control.