Figure 3(A) miR-10b miRNA recognition elements (MREs) in the 3'-UTRs of PTEN and HOXD10 genes. The coding DNA sequence (cds) and MRE are shown in red and blue, respectively. The new PTEN MRE is located 145 bp downstream of the cds, with its pairing sequence located between nucleotides 2388 and 2410 of the main PTEN transcript isoform. Below each depiction, an alignment of each mRNA and miR-10b is shown. (B) MCF-7, SKBR-3 and T47-D breast cancer cells were transfected with gain- or loss-of-function constructs/shRNAs and their respective controls (a miR-10b-OE plasmid or anti-miR-10b). MREs of the validated target HOXD10 and of the putative targets PTEN and PIK3CA were cloned into pMIR-Report, and dual luciferase reporter assays were performed. A mutated control miR-10b (MRE MUT PTEN) was also included. Bars show the mean ± SEM from 6 independent transfections. (C) PTEN and PIK3CA expression after miR-10b modulation. Left panel shows miR-10b, PTEN and PIK3CA expression by qRT-PCR in MCF-7 cells with ectopic miR-10b expression. Right panel shows miR-10b, PTEN and PIK3CA expression by qRT-PCR in MDA-MB-231 cells after antagomiR-10b transfection. Data are from three independent transient transfections performed by triplicate. (D) To further confirm PTEN targeting, we performed gain- or loss-of-function experiments in breast cancer cell lines (MCF-7, MCF-7/CD44+, SKBR-3 and T47D) by overexpressing miR-10b or inhibiting it with a miR-10b-OE plasmid or anti-miR-10b shRNAs. PTEN and miR-10b levels were measured by qRT-PCR. The log2-fold change of PTEN (left panel) and PIK3CA (right panel) mRNA levels obtained through the 2-ΔΔCT method were plotted against their respective miR-10b expression levels. Correlation analysis was performed [PTEN (r=-0.80), PI3KCA (r=0.43)]. A two-tailed Student's t test was used to compare the two groups (*p < 0.05). SEM, standard error of the mean. (E) PTEN protein modulation by miR-10b. Upper panel: PTEN protein levels in SKBR-3, and MCF-7 cells were analyzed by Western blot. GAPDH was used as a loading control with cells cultured as in (B). Lower panel: Densitometric analysis of Western blot assays performed with three biological replicates. (F) PTEN immunohistochemistry analysis of MCF-7-miR-10b-OE or control MCF-7-miR-Scr-OE tumorxenografts. (G) miR-10b overexpression does not increase mammosphere formation in PTEN-depleted cells. MCF-7-miR-Scr-OE cells (left panels) and MCF-7-miR-10b-OE (right panels) were transfected with a Dicer-substrate RNA directed toward PTEN, and after 24 h, quantitative RT-PCR assays for PTEN, SOX2, OCT4, SNAIL and Vimentin were performed. Additionally, the same cells were analyzed for mammosphere formation. The graph shows the mean and standard error of the relative increase of each marker or number of mammospheres in PTEN-depleted cells (black bars) and in cells transfected with a scrambled control (white bars) in three independent biological experiments performed by triplicate. (H) T47-D cells were transfected with antagomiR-10b, a dsi-RNA for PTEN or both and mammospheres produced as described in material and methods. Bars show mean and SEM from three independent experiments. * p=0.003 by Student´s T test, after ANOVA showing statistical differences in groups. (I) PTEN overexpression compensates for miR-10b overexpression. Left panel: MDA-468 cells were transfected with a plasmid overexpressing wild-type PTEN or a version containing a mutated MRE in its 3'-UTR, with or without a plasmid containing miR10b and PTEN mRNA expression was measured by end-point RT-PCR. Middle panel: Relative expression of stemness factor SNAIL. The plots show the relative expression from biological triplicate analyses (mean and SE shown). Right panel: Relative expression of stemness factor SOX2. The plots show the relative expression from biological triplicate analyses (mean and SE shown).