Fig 2: Experimental verification of the relationship between DNMT3A/3B and miR-200b-3p.A miR-200b-3p expression extracted from miRNA-seq results (n = 3, P = 0.008). B Relative miR-200b-3p expression in vitro when overexpression of PPARG2. *P < 0.05 vs. EV group. C Putative miR-200b-3p-binding 3′-UTR sequence of DNMT3A/3B mRNA. Mutation was generated on the DNMT3A/3B mRNA 3′-UTR sequence in the complementary site for the seed region of miR-200b-3p. The wild-type or mutant miR-200b-3p-binding DNMT3A/3B mRNA 3′-UTR sequence was cloned into pmiR luciferase reporter. D The wild-type (DNMT3A/3′-UTR-WT) and mutant (DNMT3A/3′-UTR-Mut) pmiR luciferase reporter were co-transfected into PC3 cells with miR-CON and miR-200b-3p. *P < 0.05 vs. miR-CON group. E The wild-type (DNMT3B/3′-UTR-WT) and mutant (DNMT3B/3′-UTR-Mut) pmiR luciferase reporter were co-transfected into PC3 cells with miR-CON and miR-200b-3p. *P < 0.05 vs. miR-CON group. F Relative miR-200b-3p expression in vitro transfected with miR-200b-3p mimic or miR-200b-3p inhibitor by RT-qPCR. U6 was used as an internal control. *P < 0.05 vs. miR-CON group, #P < 0.05 vs. scramble group. G, H Analysis of DNMT3A protein expression by western blotting when transfected with miR-CON, miR-200b-3p mimic, scramble, or miR-200b-3p inhibitor into PC3 cells. β-Actin was used as an internal control. *P < 0.05 vs. miR-CON group, #P < 0.05 vs. scramble group. I, J Analysis of DNMT3B protein expression by western blotting when transfected with miR-CON, miR-200b-3p mimic, scramble, or miR-200b-3p inhibitor into PC3 cells. β-Actin was used as an internal control. *P < 0.05 vs. miR-CON group, #P < 0.05 vs. scramble group.

Fig 3: miR-451a targets EPHA2 to regulate the PI3K/AKT signaling. a, the targeting mRNAs of miR-451a predicted by StarBase and analyzed using a DAVID 6.8 online bioinformatics analysis tool. Bubble plots showed 14 signaling pathways; b, gene distribution in the PI3K/AKT signaling in the KEGG database; c, EPHA2 mRNA expression in BCa cell lines T24, 5637, TCCSUP, UM-UC3 and SV-HUC-1 cells determined by RT-qPCR; d, EPHA2 protein expression in BCa cell lines T24, 5637, TCCSUP, UM-UC3 and SV-HUC-1 cells determined by western blot (full-length blots/gels are presented in Supplementary Figure 2A, B); e, EPHA2 mRNA expression in BCa cell lines after transfection determined by RT-qPCR; f, EPHA2 protein expression in BCa cell lines after transfection determined by western blot (full-length blots/gels are presented in Supplementary Figure 3A-D); g, the binding relationship between miR-451a and EPHA2 verified by dual-luciferase assays. Statistically significant differences were calculated using one-way or two-way ANOVA, followed by Tukey’s multiple comparison test. **p < 0.01 vs sh-NC or sh-DNMT3B + inhibitor NC treatment, ***p < 0.001 vs SV-HUC-1 cells

Fig 4: DNMT3B promotes miR-451a promoter methylation. a, DNMT3B expression in BCa and matched adjacent tissues in the Oncomine database; b, DNMT3B expression in normal and tumor tissues in TCGA databases predicted by the GEPIA website; c, the association between DNMT3B expression and the histological subtype of BC patients; d, the association between DNMT3B expression and the clinical stage of BC patients; e, the association between DNMT3B expression and the gender of BCa patients; f, DNMT3B mRNA expression in BCa cell lines T24, 5637, TCCSUP, UM-UC3 and SV-HUC-1 cells determined by RT-qPCR; g, DNMT3B protein expression in BCa cell lines T24, 5637, TCCSUP, UM-UC3 and SV-HUC-1 cells determined by western blot (full-length blots/gels are presented in Supplementary Figure 1A, B); h, the binding relationship between DNMT3B and miR-451a promoter in T24 and 5637 cells tested by ChIP-qPCR; i, promoter methylation in T24 cells in response to shDNMT3B (the top 3 haplotypes of high frequency were presented); j, promoter methylation in 5637 cells in response to shDNMT3B (the top 3 haplotypes of high frequency were presented); k, miR-451a expression in BCa cell lines in response to shDNMT3B determined by RT-qPCR. Data are represented as the means ± SD from three independent assays. Statistically significant differences were calculated using unpaired t-test (panel b), one-way (panel c and d) or two-way ANOVA (panel f-k), followed by Tukey’s multiple comparison test. *p < 0.05 vs tumor tissues, **p < 0.01 vs sh-NC or IgG treatment, ***p < 0.001 vs SV-HUC-1 cells

Fig 5: Schematic diagram. DNMT3B inhibits miR-451a transcription by methylation modification of the miR-451a promoter, thus activating EPHA2 and the following PI3K/AKT signaling, leading to BCa progression