![]() Total RNA (10–20 μg) was diluted with 2× RNA sample loading buffer (95% formamide, 18 mmol/L EDTA, 0.25% SDS, 0.25% xylene cyanol, and 0.25% bromophenol blue), denatured at 95☌ for 5 minutes, and separated on 15% polyacrylamide gels containing 8 mol/L urea. Average values of replicate spots of each miRNA were background subtracted, quantile normalized, and subjected to further analysis (GEO accession: GSE53504). Hybridization signals were quantified using the GenePix 6.0 software (Axon Instruments). The hybridized chips were washed and processed to detect biotin-containing transcripts by streptavidin-Alexa647 conjugate and scanned on an Axon 4000B microarray scanner (Axon Instruments). Hybridization of biotin-labeled cDNA was performed on the new Ohio State University custom miRNA microarray chip (OSU_CCC version 3.0), which contains approximately 1,100 miRNA probes, including 326 human and 249 mouse miRNA genes, spotted in duplicates. In brief, 5 μg of total RNA from each sample was reverse transcribed using biotin end-labeled random-octamer oligonucleotide primers. RNA labeling and hybridization to miRNA microarray chips was performed as described ( 24). Glioma cells were starved 48 hours before total RNA extraction using TRIzol (Life Technologies). Collectively, this broad spectrum of biologic activities provides a compelling rationale for the molecular targeting of EGFR in glioblastoma. ΔEGFR confers a variety of biologic effects upon its expression, including resistance to radiation ( 4) and chemotherapeutic agents ( 5), promotion of tumor cell motility and invasion ( 6), enhancement of tumorigenicity in vivo ( 7), maintenance of glioblastoma growth ( 8), and heterogeneity ( 9). The most common EGFR mutant, ΔEGFR (also known as EGFRvIII and de2-7), is generated from an in-frame 801-bp deletion of exons 2–7 ( 3), and is constitutively active and present in a high proportion of glioblastomas with EGFR amplification ( 2). Molecular analyses have shown that 40% to 50% of primary glioblastomas have EGFR amplification, overexpression, and/or mutations ( 2). Glioblastomas infiltrate normal brain parenchyma, display a high degree of cellular and genetic intratumoral heterogeneity, and exhibit limited responses to conventional therapies ( 1). Collectively, these data suggest a novel regulatory mechanism by which ΔEGFR suppression of miR-9 upregulates FOXP1 to increase tumorigenicity. The significance of these findings is underscored by our finding that high FOXP1 expression predicts poor survival in a cohort of 131 patients with glioblastoma. FOXP1 was sufficient to increase tumor growth in the absence of oncogenic ΔEGFR signaling. Silencing of FOXP1, a miR-9 target, inhibits ΔEGFR-dependent tumor growth and, conversely, de-repression of FOXP1, as a consequence of miR-9 inhibition, increases tumorigenicity. ![]() ![]() Correspondingly, expression of miR-9 antagonizes the tumor growth advantage conferred by ΔEGFR. Here, we report that ΔEGFR specifically suppresses one such microRNA, namely miR-9, through the Ras/PI3K/AKT axis that it is known to activate. This range of activities suggested to us that ΔEGFR might exert its influence through pleiotropic effectors, and we hypothesized that microRNAs might serve such a function. The EGF receptor (EGFR) is amplified and mutated in glioblastoma, in which its common mutation (ΔEGFR, also called EGFRvIII) has a variety of activities that promote growth and inhibit death, thereby conferring a strong tumor-enhancing effect. ![]()
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