Fig. 1

Expression and epigenetic regulation of miR-212-3p in group 3 MB tumors. A MiR-212 expression analysis by RNA Sequencing using (i) a publicly available MB dataset (NC n = 14; MB n = 15; Drusco et al., GSE62381) and (ii) a local MB cohort (adult NC n = 4; pediatric NC n = 10; SHH n = 5; group 3 MB n = 9; group 4 MB n = 13; Kanchan et al., GSE148390). B Results validated in local MB cohort by RT-PCR (NC n = 10; G3MB n = 10; G4MB n = 12). C RT-PCR analysis of miR-212 expression in classic MB cell lines (group 3: D341, D425, HDMB03; group 3/4: D283; SHH: Daoy) compared to normal human astrocytes (NHA). D In silico expression of HDACs and EZH2 to investigate epigenetic modulation of miR-212 (NC n = 10; group 3 MB n = 7; Kanchan et al., GSE148390). E ChIP-qRT-PCR analysis in group 3 MB cells with deregulated histone modifications in the promoter region of miR-212-3p. ChIP grade histone mark antibodies to H3K27me3, H3K9me2 and H3K9Ac used; IgG antibody as negative control; Daoy cells with high intrinsic miR-212-3p expression served as an additional control. F MiR-212-3p expression restoration after treatment with pan-HDAC inhibitors (TSA, 100 nM; Belinostat, 1 µM; and Vorinostat, 1 µM) and with siRNA-EZH2 (20 nM) in HDMB03 cells. Increase in acetylated α-tubulin levels demonstrated pan-HDAC inhibitor activity in HDMB03 cells. β-actin served as an internal control. Data presented as mean ± SD from experiments done in triplicate and analyzed using Mann–Whitney U test (A and D) or Student’s t-test (B, C, E and F); *p < 0.05, **p < 0.01, ***p < 0.001. NC, normal cerebellum; MB, medulloblastoma; G3MB, group 3 medulloblastoma; G4MB, group 4 medulloblastoma