Identification of Dysregulated microRNAs in Glioblastoma Stem-like Cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite multimodal therapy, median survival is poor at 12-15 months. At the molecular level, radio-/chemoresistance and resulting tumor progression are attributed to a small fraction of tumor cells, termed glioblastoma stem-like cells (GSCs). These CD133-expressing, self-renewing cells display the properties of multi-lineage differentiation, resulting in the heterogenous composition of GBM. MicroRNAs (miRNAs) as regulators of gene expression at the post-transcriptional level can alter many pathways pivotal to cancer stem cell fate. This study explored changes in the miRNA expression profiles in patient-derived GSCs altered on differentiation into glial fiber acid protein (GFAP)-expressing, astrocytic tumor cells using a polymerase chain reaction (PCR) array. Initially, 22 miRNAs showed higher expression in GSCs and 9 miRNAs in differentiated cells. The two most downregulated miRNAs in differentiated GSCs were miR-17-5p and miR-425-5p, whilst the most upregulated miRNAs were miR-223-3p and let-7-5p. Among those, miR-425-5p showed the highest consistency in an upregulation in all three GSCs. By transfection of a 425-5p miRNA mimic, we demonstrated downregulation of the GFAP protein in differentiated patient-derived GBM cells, providing potential evidence for direct regulation of miRNAs in the GSC/GBM cell transition.

The Metalloprotease-Disintegrin ADAM8 Alters the Tumor Suppressor miR-181a-5p Expression Profile in Glioblastoma Thereby Contributing to Its Aggressiveness.

Glioblastoma (GBM) as the most common and aggressive brain tumor is characterized by genetic heterogeneity, invasiveness, radio-/chemoresistance, and occurrence of GBM stem-like cells. The metalloprotease-disintegrin ADAM8 is highly expressed in GBM tumor and immune cells and correlates with poor survival. In GBM, ADAM8 affects intracellular kinase signaling and increases expression levels of osteopontin/SPP1 and matrix metalloproteinase 9 (MMP9) by an unknown mechanism. Here we explored whether microRNA (miRNA) expression levels could be regulators of MMP9 expression in GBM cells expressing ADAM8. Initially, we identified several miRNAs as dysregulated in ADAM8-deficient U87 GBM cells. Among these, the tumor suppressor miR-181a-5p was significantly upregulated in ADAM8 knockout clones. By inhibiting kinase signaling, we found that ADAM8 downregulates expression of miR-181a-5p via activation of signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) signaling suggesting an ADAM8-dependent silencing of miR-181a-5p. In turn, mimic miR-181a-5p transfection caused decreased cell proliferation and lower MMP9 expression in GBM cells. Furthermore, miR-181a-5p was detected in GBM cell-derived extracellular vesicles (EVs) as well as patient serum-derived EVs. We identified miR-181a-5p downregulating MMP9 expression via targeting the MAPK pathway. Analysis of patient tissue samples (n=22) revealed that in GBM, miR-181a-5p is strongly downregulated compared to ADAM8 and MMP9 mRNA expression, even in localized tumor areas. Taken together, we provide evidence for a functional axis involving ADAM8/miR-181a-5p/MAPK/MMP9 in GBM tumor cells.