Skip to main content
. 2015 Jul 16;6(7):e1823. doi: 10.1038/cddis.2015.192

Figure 2.

Figure 2

miR-205-5p regulates ERBB receptors expression in BCSCs. (a) qRT-PCR quantification of miR-205-5p expression in BCSC#1, BCSC#2 and BCSC#3 and SDAC differentiated for 3.5 and 7 days. miR-205-5p is downregulated during differentiation in all three stem cell lines tested. Data represent mean±S.D. of three different experiments analyzed in triplicate. (b and c) miR-205-5p regulates ERBB2 and EGFR expression. qRT-PCR and western blotting analysis of EGFR and ERBB2 expression levels in BCSC#1 infected with miR-205-5p silencing lentivector (shmiR-205-5p). miR-205 knockdown results in EGFR and ErbB2 upregulation both at the mRNA and protein levels. ZEB-1, a well-established miR-205 target, was used as a control to further confirm functional miR-205 silencing. (d and e) miR-205-5p overexpression results in ERBB receptors downregulation. qRT-PCR and western blot analysis of EGFR, ERBB2 and Zeb-1 expression levels in BCSC#1 infected with PremiR-205 lentivector. All qRT-PCR data represent mean±S.D. of three different experiments analyzed in triplicate. (f) miR-205-5p directly targets ERBB2 at 3'-UTR. Schematic model of the predicted ERBB2 3'-UTR binding site for miR-205-5p and alignment of the seed region with both wild-type and mutated ERBB2 3'-UTR (left). On the right, relative luciferase activity is shown. SKBR-3 cells were co-transfected for 24 h with pGL3-ERBB2 3'-UTR luciferase construct (WT or Mut 3'-UTR), premiR 205 construct or a control vector (CTR). The results represent mean±S.D. of three different experiments analyzed in triplicate. (g) EGFR is not a direct target of miR-205-5p. Representation of the interaction between miR-205-5p and the putative binding site on the wild-type EGFR 3'-UTR (left) and relative luciferase activity (right) in SKBR-3 cells transfected with premiR 205 construct or a control vector (CTR) at the indicated time points (hours)