Abstract
Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.
Highlights
Glioblastoma multiforme (GBM) is the highest grade (IV) astrocytoma and the most common glioma, accounting for ~40% of all primary brain tumors of the central nervous system (CNS) [1]
Characterization of the neurosphere cultures obtained from surgical GBM samples Five cell lines derived from GBM biopsies were cultured in a GBM-initiating cells (GICs)-propagating medium, where they formed typical spherical structures in suspension known as neurospheres (NS) (Figure 1A, left panels) expressing the neural progenitor cell marker Nestin (Figure 1B-C)
A statistically significant increase in the mRNA levels of GFAP and/or TUJ1 was observed (Figure 1B)
Summary
Glioblastoma multiforme (GBM) is the highest grade (IV) astrocytoma and the most common glioma, accounting for ~40% of all primary brain tumors of the central nervous system (CNS) [1]. GBM is one of the most aggressive tumors. Patients usually have a median overall survival of 12-15 months, due to the high rate of tumor recurrence despite surgical tumor removal and radio-chemotherapy, which highlights the need for more effective therapies [2]. It has been proposed that gliomagenesis initiates in adult neural stem cells or neural precursors that undergo transformation into GBM-initiating cells (GICs), which display a stem cell-like behavior [3]. GICs are able to self-renew, express stem cell markers such as CD133 and Nestin, and can generate and propagate tumors in immunodeficient mice [3,4,5].
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