MicroRNA-199b-5p Impairs Cancer Stem Cells through Negative Regulation of HES1 in Medulloblastoma
Figure 3
The effects of miR-199b-5p over-expression are reversed by HES1 transfection.
A) Representative Western blot and densitometric analysis showing that the rescue of HES1 expression by over-expression of HES1 cDNA in the stable 199bSC1 clone restores cyclin D1 expression. B) Proliferation assay by 3-4,5-dimethylthiazol-2-yl-5-3-carboxymethoxyphenyl-2-4-sulfophenyl-2H-tetrazolium salt (MTS) showing increased proliferation rate of the stable 199bSC1 clone with HES1 expression (dark blue squares), compared to the stable 199bSC1 clone alone (light blue diamonds). The data shown are means±SD from two independent experiments, each carried out in triplicate. C) Rapresentative mRNA expression of differentiation (e.g. MASH1, MATH3 and NEUROGENIN 2) and proliferation (e.g. c-MYC, CYCLIN D1) markers upon cell-rescue with HES1 in the stable 199bSC1 clone, comparing with wild-type cells, as revealed by real-time PCR. The expression of GABRA 6 and MAP2 are down-regulated in the rescue experiment. D) Representative FACS analysis of the HES1-expressing stable 199bSC1 clone shows an increase in the fraction of cells in S phase, and a decrease in G1, with respect to the stable 199bSC1 clone alone. E–H) The effect of miR-199b-5p expression on the SP of Daoy cells is reversed by HES1 re-expression. 199bSC1 stable clone was transfected with HES1 cDNA and a GFP-coding plasmid, then treated after 48 hours with Hoechst and verapamil (E–F) or Hoechst alone (G–H) and analyzed by FACS. The GFP-positive cells (P5 gate), panel E and G, were analysed for the presence of dye-excluding-cells (F–H, P6 gate). The untransfected GFP negative cells (P3 gate), were not analised.