Fig. 3. Identification of SUV420H2 as a direct target gene of miR-29a in breast cancer cells.

a Left: Venn diagram analysis of three independent databases reveals four possible targets of miR-29a. Right: schematic description of the hypothetical duplexes formed by the interactions between the binding sites in the SUV420H2 3′-UTR and miR-29a. The predicted free energy value of each 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, c SUV420H2 protein (b) and mRNA (c) levels in MCF-7 cells, MCF-7 spheroid cells and CD44+/CD24− MCF-7 cells. d, e SUV420H2 protein (d) and mRNA (e) levels in 12 pairs of human breast cancer tissues (Cancer) and corresponding distal non-cancerous tissues (Normal). f Pearson’s correlation scatter plot of the fold change of miR-29a and SUV420H2 protein in human breast cancer tissues. g Pearson’s correlation scatter plot of the fold change of miR-29a and SUV420H2 mRNA in human breast cancer tissues. h, i SUV420H2 protein (h) and mRNA (i) levels in MCF-7 cells and MDA-MB-231 cells. j Direct recognition of the SUV420H2 3′-UTR by miR-29a. Firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-29a-binding sites in the SUV420H2 3′-UTR were co-transfected into MCF-7 cells with either the control mimic, control inhibitor, miR-29a mimic or miR-29a inhibitor. Twenty-four hours post-transfection, the cells were assayed using a luciferase assay kit. The results were calculated as the ratio of firefly luciferase activity normalized to the control cells. k, l SUV420H2 protein (i) and mRNA (j) levels in MCF-7 cells transfected with scrambled negative control RNA, miR-29a mimic, or miR-29a inhibitor. *P < 0.05; **P < 0.01