Figure 4.

miR-205-5p regulates p63 expression in BCSCs. (a) miR-205-5p regulates p63 levels. qRT-PCR (left) and western blotting analysis (right) of p63 expression levels in BCSC#1 infected with miR-205-5p silencing lentivector (shmiR-205-5p). (b) qRT-PCR (left) and western blotting (right) of p63 expression levels in BCSC#1 infected with Premir-205 lentivector. miR-205-5p overexpression results in p63 downregulation mainly at protein levels. (c) miR-205-5p directly targets p63-3'-UTR. On the left, putative miR-205-5p binding site on p63 wild-type 3'-UTR and alignment of the seed sequence with both WT and mutated p63 3'-UTR. On the right, SKBR-3 cells were co-transfected for 48 h with pGL3-p63 3'-UTR luciferase construct (WT or Mut 3'-UTR), premiR 205 construct or a control vector (CTR). Cloning p63 3'-UTR WT, and not the mutated one, into a luciferase reporter gene leads to diminished luciferase activity in the presence of Premir-205. (d) p63 regulates miR-205-5p expression. qRT-PCR of miR-205-5p expression levels in BCSC#1 infected with shp63 lentivector (shp63) or a control vector (plentilox). (e) qRT-PCR of miR-205-5p expression levels upon Tap63 or ΔNp63 overexpression in BCSC#1. ΔNp63 overexpression results in miR-205-5p upregulation. (f) miR-205-5p downregulation re-sensitize BCSCs to Lapatinib treatment. Percentage of cell growth of BCSC#1, BCSC#2 and BCSC#3 infected with shmiR-205-5p lentivector (shmiR-205-5p) or a control vector (CTR) and treated or untreated with 0.5 μM Lapatinib for 6 days