Fig. 6: CMYC promotes HNF1A-AS1 expression by directly binding to the promoter of HNF1A-AS1.

A Transcriptional activity analysis of the potential HNF1A-AS1 promoter fragments in MKN-45 cells (n = 3). B Relative luciferase activity assay showed that CMYC observably increased promoter activities (n = 3). C qRT-PCR assay indicated CMYC promoted the expression level of HNF1A-AS1 in GC cells (n = 3). D, E Relative luciferase activity of the HNF1A-AS1 promoter was decreased when the three presumed HNF1A-AS1 binding site was mutated (n = 3). F, G ChIP-qPCR analysis indicated higher fold enrichment of promoter amplicons of CMYC in anti-CMYC antibody group than that of IgG group in GC cells, indicating that CMYC could directly bind to HNF1A-AS1 promoter (n = 3). M1, M2 and M3 respectively represent primer that covered the CMYC-binding sites. H, I Gastric cancer tissue samples were immunohistochemically stained for CMYC and β-catenin for expression scoring. Correlation analysis was performed with HNF1A-AS1 expression levels (n = 26). J Western blotting analyses of stemness-related gene expression in mouse xenograft tumors. K Spatial localization of β-catenin in mouse xenograft tumor cells determined by Western blotting. L Representative images of IHC for β-catenin and CMYC in tumors from xenograft mice. Original magnification, ×400. M Relative quantification of mRNA expression of β-catenin and CMYC in tumors from xenograft mice (n = 3). Data are representative as the mean ± SD. One-way ANOVA with Tukey’s multiple-comparison test (A, B, D, E), two-tailed unpaired Student’s t-test (C, F, G, M), Spearman’s correlation (H, I). *P < 0.05; **P < 0.01; ***P < 0.001.