Figure 3

Inhibition of miR-10b suppresses angiogenesis by U87-2M1 glioma cells. (a) In vivo effects. Intracranial tumors produced from miR-10b decoy or control decoy vector-transduced U87-2M1 cells were sectioned and immunostained using antibody against CD31. Bars equal 200 μm. (b) In vitro effects. Angiogenic potential of U87-2M1 cells were determined by an in vitro endothelial cell tube formation assay. Conditioned medium from U87-2M1 cells transduced with control or miR-10b decoy vector were added to human vascular endothelial cells plated on Matrigel. Bars equal 200 μm. (c) Downregulation of expression of pro-angiogenic proteins in U87-2M1 cells by miR-10b decoy as demonstrated in western blotting analysis. Numbers indicate relative protein expression in miR-10b decoy-treated U87-2M1 cells compared with control decoy-transduced U87-2M1 cells after normalization to beta-actin levels. Blots shown are representative of biological triplicates. (d) A panel of 13 angiogenic genes downregulated by miR-10b silencing is frequently overexpressed in the Mesenchymal subtype of GBM. Most of the genes in this panel are less frequently overexpressed in the Classical subtype of GBM, and frequently underexpressed in the ‘Neural’ and ‘Proneural’ subtypes of GBM. Oncoprints involving the panel of 13 angiogenic genes in the Mesenchymal, Classical, Neural and Proneural subtypes of GBM were derived from the cBio Cancer Genomics Portal.¹⁴ In each oncoprint, individual genes are represented as rows and individual patients or cases are represented as columns. The stratification of the patients and cases from TCGA GBM was done according to the molecular classification defined by Verhaak et al.² Red-outlined boxes indicate mRNA overexpression and red-filled boxes indicate amplification. Blue-outlined boxes indicate mRNA underexpression and blue-filled boxes indicate homozygous deletions. Green boxes indicate presence of gene mutation