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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

From: Methylation-mediated silencing of miR-133a-3p promotes breast cancer cell migration and stemness via miR-133a-3p/MAML1/DNMT3A positive feedback loop

Fig. 4

Identification of MAML1 as a direct target gene of miR-133a-3p in breast cancer cells. a Left: Venn diagram analysis of four independent databases reveals 96 possible targets of miR-133a-3p. Right: Schematic description of the hypothetical duplexes formed by the interactions between the binding sites in the MAML1 3′-UTR and miR-133a-3p. The predicted free energy value of the hybrid is indicated. The seed recognition sites are denoted, and all nucleotides in these regions are highly conserved across species, including human, mouse, and rat. b Luciferase activity in MCF-7 cells co-transfected with a luciferase reporter containing either MAML1-WT or MAML1-MUT (miR-133a-3p-binding sequence mutated), and mimics NC, miR-133a-3p mimics, inhibitor NC or miR-133a-3p inhibitor. Data are presented as the relative ratio of renilla luciferase activity and firefly luciferase activity. c Relative enrichment of MAML1 mRNA and miR-133a-3p associated with AGO2 in MCF-7 (left) and MDA-MB-231 (right) cells detected by anti-AGO2 RIP (non-specific IgG as negative control). d, e MAML1 mRNA (d) and protein (e) levels in MCF-7 cells and MDA-MB-231 cells transfected with mimics NC, miR-133a-3p mimics, inhibitor NC, or miR-133a-3p inhibitor. f, g MAML1 protein levels (f) and Pearson’s correlation scatter plot of the fold change of miR-133a-3p and MAML1 protein (g) in 12 pairs of human breast cancer tissues (BRCA) and corresponding distal non-cancerous tissues (NAT). h Overall survival analysis of breast cancer patients based on MAML1 expression (n = 3951, log-rank test). Data was analyzed using Kaplan Meier Plotter (www.kmplot.com). i, j MAML1 protein levels (i) and Pearson’s correlation scatter plot of the fold change of miR-133a-3p and MAML1 protein (j) in different breast cancer cells and normal breast cells. **P < 0.01; ***P < 0.001

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