Fig. 5

miR-10b regulates alternative splicing of CDC42, thereby controlling its total levels via U6 regulation. a Alternative splicing events induced by miR-10b inhibitor and U6 ASO #1, as determined by RNAseq (n = 3). Venn diagrams indicate the numbers of exon skipping (SE) and alternative 3′ splice sites (A3SS) modulation, in the indicated conditions. b Schematic illustration of two major CDC42 mRNA isoforms, with alternative exons encoding alternative 5′ and 3′ UTRs (left panel). Expression analysis of CDC42 isoforms in the normal brain (n = 1141), low grade glioma (LGG) (n = 509) and GBM (n = 153) in TCGA database demonstrates that ENST00000344548 (CDC42-iso2) is the pathologic variant associated with glioma progression, whereas ENST00000315554 (CDC42-iso1) is present at similarly low levels in the normal brain, LGG, and GBM (right panel). c qRT-PCR analysis of CDC42-iso1 or iso2 mRNAs in glioma cells and GSCs transfected with either U6 ASOs, or miR-10b inhibitor or mimic. The results are expressed as the fold-changes relative to the corresponding control groups (mean ± SD, n = 3). P values were calculated using two-tail unpaired t-test. d Western blot analysis of the indicated CDC42 forms in the cells transfected with either U6 ASO, or miR-10b inhibitor or mimic. e Western blot analysis of the cells transfected with either selective siRNA targeting iso2 (siCDC42-iso2) or both CDC42 isoforms (siCDC42-total) demonstrates that KD of iso2 is sufficient for reducing total CDC42 levels. f Analysis of GSC spheroids transfected with either siCDC42-iso2 or siCDC42-total demonstrates that CDC42 KD reduces GSC growth. The number and size of GSC spheres have been monitored using Image J at day 7 after transfection (mean ± SD, n = 3, 8 images per cultured analyzed). P values were calculated using two-tail unpaired t-test. g Analysis of glioma LN229 cells transfected with either siCDC42-iso2 or siCDC42-total demonstrates that CDC42 KD reduces glioma growth. Cell viability was monitored 72 h after transfection by WST-1 assay (mean ± SD, n = 3). P values were calculated using two-tail unpaired t-test. h Representative images of LN229 cells immuno-stained for Ki-67 (green) and DAPI staining (blue) and quantification of the Ki67⁺ cells (mean ± SD, n = 3). P values were calculated using two-tail unpaired t-test. i Schematic illustration of alternative splicing of CDC42, regulated by miR-10b via its binding to U6 snRNA. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, no significance