miR-33a promotes glioma-initiating cell self-renewal via PKA and NOTCH pathways
Hui Wang, … , Huibo Wang, Xiao-Fan Wang
Hui Wang, … , Huibo Wang, Xiao-Fan Wang
Published September 9, 2014
Citation Information: J Clin Invest. 2014;124(10):4489-4502. https://doi.org/10.1172/JCI75284.
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Research Article Oncology

miR-33a promotes glioma-initiating cell self-renewal via PKA and NOTCH pathways

Abstract

Glioblastoma (GBM) is the most common and lethal brain tumor in adults. Glioma-initiating cells (GICs) are stem-like cells that have been implicated in glioblastoma progression and recurrence; however, the distinct properties of GICs and non-GICs within GBM tumors are largely uncharacterized. Here, we evaluated stem cell–associated microRNA (miR) expression in GICs from GBM patients and GICs derived from xenografted human glioma cell lines and determined that miR-33a promotes GIC growth and self-renewal. Moreover, evaluation of a GBM tissue array revealed that higher miR-33a expression was associated with poor prognosis of GBM patients. Antagonizing miR-33a function in GICs reduced self-renewal and tumor progression in immune-compromised mice, whereas overexpression of miR-33a in non-GICs promoted the display of features associated with GICs. We identified the mRNAs encoding phosphodiesterase 8A (PDE8A) and UV radiation resistance–associated gene (UVRAG) as direct miR-33a targets. PDE8A and UVRAG negatively regulated the cAMP/PKA and NOTCH pathways, respectively; therefore, miR-33a–dependent reduction of these proteins promoted growth and self-renewal of GICs by enhancing PKA and NOTCH activity. Furthermore, in GBM specimens, there was an inverse correlation between the expression levels of miR-33a and PDE8A and UVRAG expression. These findings reveal a miR-33a–centered signaling network that promotes GIC maintenance and has potential as a therapeutic target for GBM treatment.

Authors

Hui Wang, Tao Sun, Jing Hu, Rui Zhang, Yanhua Rao, Shuai Wang, Rui Chen, Roger E. McLendon, Allan H. Friedman, Stephen T. Keir, Darell D. Bigner, Qi-Jing Li, Huibo Wang, Xiao-Fan Wang

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Figure 3

PDE8A and UVRAG are direct targets of miR-33a in glioma cells.

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PDE8A and UVRAG are direct targets of miR-33a in glioma cells. (A) An il...
(A) An illustration of strategies to identify direct targets of miR-33a. (B) RNA-Ago2 immunoprecipitation (IP) using lysates from D456MG CD133– cells expressing the vector control or miR-33a as indicated. β-Actin was used as the loading control. (C) qRT-PCR analysis was performed to measure miR-33a levels incorporated into RISC in miR-33a–overexpressing cells compared with those of the control. RNU6 was used as an internal control. (D) qRT-PCR analysis was performed to measure the levels of indicated mRNAs incorporated into RISC derived from miR-33a–overexpressing or vector control cells. ACTB was used as an internal control. (E) Immunoblotting was performed to show PDE8A and UVRAG protein levels in D456MG and 11-0040 cells as indicated, with repression (miRZIP) or sponge as marked) or overexpression of miR-33a. Intensities of bands were quantified using Image J and γ-tubulin was used as internal control to calculate the fold-change. (F) Luciferase activity of the reporter construct containing the wild type or miR-33a–binding mutant 3′ UTR of PDE8A or UVRAG was measured after cotransfection with control, or 0.5 μg or 1 μg miR-33a–expressing construct, respectively, in 293FT cells. *P < 0.05; **P < 0.01 compared with control group.