Fig. 3.

miR-10-5p is activated by SOX2 and correlates with the stem cell and tumor phenotype of GBM. a Venn diagram showing the intersection of TET2-targeting miRNAs predicted using three different algorithms (top panel). List of high-confidence miRNAs predicted to target TET2 (bottom panel). b qRT-PCR to measure the expression of pre-cursor miRNAs predicted to inhibit TET2 in GSCs expressing transgenic SOX2. Western blot showing SOX2 protein levels in GSCs expressing transgenic Sox2 (inset). c qRT-PCR to measure miR-10b-5p expression in GBM neurospheres lines and primary GBM neurosphere isolates compared to non-tumorigenic glial progenitors (normal). d Correlation between Sox2 and miR-10b-5p expression in primary GSC isolates. e Sox2 binding sites on the human miR-10b-5p promoter, arrows indicate primer sites used for PCR analyses (top panel). DNA purified from chromatin immunoprecipitation was analyzed by qRT-PCR using primer pairs designed to amplify fragments containing Sox2 (bottom panels). f 293T cells were co-transfected with a luciferase reporter construct spanning the miR-10b-5p putative promoter containing the SOX2 binding sites and GFP, OCT4, or SOX2 and luciferase activity was measured 2 days after transfection. g GSC isolates expressing exogenous SOX2 were transfected with the luciferase reporter construct spanning the miR-10b-5p putative promoter containing the SOX2 binding sites and luciferase activity was measured 3 days after transfection. h GBM1A and GBM1B neurospheres were transfected with luciferase reporter construct covering the miR-10b-5p putative promoter containing the SOX2 binding sites and forced to differentiate. Luciferase activity was measured 3 days after differentiation. i miR-10b-5p levels in normal brain compared to GBM subtypes. j Kaplan–Meier survival curves comparing GBM patient survival based on miR-10b-5p expression. miR-10b-5p expression, and patient survival data were retrieved from the TCGA database using the BETASTASIS portal (http://www.betastasis.com). One-way ANOVA with Tuckey’s post hoc test was used to calculate statistical significance in f and i. Statistical significance was calculated using Student’s t-test in b, c, d, f, g, and h. Data are presented as mean ± SD. *p < 0.05