Fig 1: Six1 reintroduction rescues the TMZ‐resistant effect of miR‐155‐3p. A, Cell viability of A172 cells transfected with miR‐con or miR‐155‐3p with or without Six1 overexpression was treated with increasing dose of TMZ. B, Cell viability of U87 cells transfected with miR‐con or miR‐155‐3p with or without Six1 overexpression was treated with increasing dose of TMZ. C, Colony formation assay of A172 cells transfected with miR‐con or miR‐155‐3p with or without Six1 overexpression was treated with increasing dose of TMZ. D, Colony formation assay of U87 cells transfected with miR‐con or miR‐155‐3p with or without Six1 overexpression was treated with increasing dose of TMZ. E, Western blotting of cleaved caspase 3 of A172 cells transfected with miR‐con or miR‐155‐3p with or without the Six1 overexpression treated with 100 μmol/L TMZ for 48 h. F, Western blotting of cleaved caspase 3 of U87 cells transfected with miR‐con or miR‐155‐3p with or without the Six1 overexpression treated with 100 μmol/L TMZ for 48 h. Results were expressed as means ± SD of 3 independent experiments. **P < .01
Fig 2: SIX1 was a direct target of sf-RON/β-catenin to regulate glycolysis in gastric cancer cells. a GSEA showed high enrichment of the SIX1 pathway from RNA sequencing data. b Western blotting showed that the expression level of SIX1 was enhanced in sf-RON-overexpressing gastric cancer cells, compared with RON-overexpressing and negative control cells. c, d Silencing of β-catenin reduced the expression level of SIX1 in sf-RON-overexpressing and RON-overexpressing gastric cancer cells detected by western blotting and qRT-PCR assay. e SIX1 rescued the silencing of β-catenin on the expression of GLUT1 and LDHA in gastric cells detected by western blotting assay. f Luciferase reporter assay was used to detect the regulation of β-catenin on the promoter activity of SIX1. ** P < 0.01. g, h PCR results of ChIP analysis showed that β-catenin bound to the SIX1 gene promoter region and the map of β-catenin binding sites in the promoter region of SIX1. i Luciferase reporter assay was used for the detection of β-catenin mutant sites in the promoter region of SIX1. ** P < 0.01. j Luciferase reporter assay showed that silencing of SIX reduced the promoter activity of GLUT1 and LDHA. ** P < 0.01
Fig 3: The associations among the expression of sf-RON, RON, β-catenin and SIX1, and survival of gastric cancer patients. a Representative images of biopsies containing the expression of β-catenin and SIX1 in gastric cancer patient tissues with low and high expression of sf-RON and RON (× 400). b, c Correlation of sf-RON and RON expression with β-catenin and SIX1. d–g Kaplan-Meier OS survival curves (log-rank tests) and PFS survival curves (log-rank tests) of patients with varying sf-RON, RON, β-catenin, and SIX1 expression. h Schematic model showing the role of sf-RON/β-catenin/SIX1 signaling axis in the regulation of cell proliferation and glucose metabolism in the GC cells
Fig 4: sf-RON promoted tumor growth by regulating β-catenin/SIX1 signaling in vivo. a Representative image of nude mice bearing tumors formed by sf-RON, sf-RON/β-catenin KD (Knockdown) and their control cells. b The average tumor volume of nude mice bearing tumors formed by sf-RON, sf-RON/β-catenin KD and their control cells. ** P < 0.01. c The average tumor weight of nude mice bearing tumors formed by sf-RON, sf-RON/β-catenin KD, and their control cells. d Representative image of PET-CT was used for the detection of glucose uptake in sf-RON, sf-RON/β-catenin KD xenografts, and their controls. e Average SUVmax values of nude mice bearing tumors. ** P < 0.01. f Immunohistochemical staining of β-catenin, SIX1, GLUT1, and LDHA in the sf-RON, sf-RON/β-catenin KD, and control tissues (magnification × 200)
Fig 5: miR‐155‐3p targeting Six1 in glioma. A, The predicted base pairing in miR‐155‐3p and Six1 from TargetScan. B, Validation of targeting relation between miR‐155‐3p and Six1 through dual‐luciferase reporter assay in A172 cells. C, Validation of targeting relation between miR‐155‐3p and Six1 through dual‐luciferase reporter assay in U87 cells. D, Western blotting of Six1 in A172 and U87 cells transfected with indicated miR‐155‐3p or anti‐miR‐155‐3p. E, Western blotting of Six1 in A172 cells transfected with miR‐155‐3p with or without Six1 overexpression plasmid. F, The mRNA level of Six1 was higher in glioma cell lines than that in normal cell line NHAs. G, Relative mRNA level of Six1 in glioma tissues was analysed by real‐time PCR. H, Spearman correlation analysis of Six1 and miR‐155‐3p level in glioma tissues. Results were expressed as means ± SD of 3 independent experiments. **P < .01
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