Fig. 3: Fra-1 and HMGA2 promoter interaction regulates HMGA2 expression and induces M2 polarization in macrophages.

A, B Prediction of potential binding sites between Fra-1 and the HMGA2 promoter regions using the JASPAR database. C, D Chromatin Immunoprecipitation followed by quantitative PCR (ChIP-qPCR) assay was conducted to detect the binding of Fra-1 to HMGA2 promoter sites in gastric cancer cells. “IP” indicates enrichment with a Fra-1 antibody; “IgG” indicates enrichment with an IgG antibody. The p-value was calculated through unpaired t-test correction. E, F Gastric cancer cells were transfected with HMGA2 wild-type (HMGA2-WT) or mutant (HMGA2-MT) dual luciferase reporter gene plasmids after Fra-1 overexpression. Luciferase activity was measured using a dual luciferase reporter gene detection kit after a 48-h incubation. Multiple comparisons were analyzed using ANOVA, and two groups were compared by Wilcoxon rank sum test. G–J The supernatant from gastric cancer cells overexpressing HMGA2 was used for co-culture with M0 macrophages. M0 macrophages were labeled with CD11b, M1 macrophages with CD86, and M2 macrophages with CD163. Flow cytometry was employed to determine the polarization ratio of M1 and M2 macrophages. The p-value was calculated through unpaired t-test correction. K–Q The effect of HMGA2 expression in gastric cancer cells on the secretion of M2 macrophage-related cytokines TGF-β, IL-10, and Arg-1 was assessed using an ELISA detection kit following co-culture with M0 macrophages. The p-value was calculated through unpaired t-test correction. R, I The supernatant from gastric cancer cells overexpressing HMGA2 was used for co-culture with M0 macrophages. RT-qPCR was used to assess the impact of HMGA2 expression on the mRNA levels of M2 macrophage-related cytokines TGF-β, IL-10, and Arg-1 in gastric cancer cells. The p-value was calculated through unpaired t-test correction. S, T Gastric cancer cell culture supernatants overexpressing Fra-1, and those overexpressing Fra-1 with simultaneous HMGA2 knockdown, were used for co-culture with M0 macrophages labeled with CD11b and M2 macrophages labeled with CD163. Flow cytometry was used to determine the polarization ratio of M2 macrophages. Multiple comparisons were analyzed using ANOVA, and two groups were compared by Wilcoxon rank sum test. U, V M0 macrophages were co-cultured with the supernatant of gastric cancer cells overexpressing Fra-1 and those overexpressing Fra-1 while knocking down HMGA2. RT-qPCR experiments were conducted to evaluate the effect of Fra-1-regulated HMGA2 expression on the mRNA levels of M2 macrophage-related cytokines TGF-β, IL-10, and Arg-1 in gastric cancer cells. Multiple comparisons were analyzed using ANOVA, and two groups were compared by the Wilcoxon rank sum test. All experiments were independently repeated three or more times, and the data presented are from representative individual experiments. “ns” indicates no significant difference; “*” indicates p < 0.05; “**” indicates p < 0.01; “***” indicates p < 0.001; “****” indicates p < 0.0001.