Propolis Extract Regulate microRNA Expression in Glioblastoma and Brain Cancer Stem Cells.

BACKGROUND Grade IV gliomas are classified as glioblastoma (GBM), which is the most malignant brain cancer type. Various genetic and epigenetic mechanisms play a role in the initiation and progression of GBM. MicroRNAs (miRNAs) are small, non-coding RNA molecules that are the main epigenetic regulatory RNA class. They play variable roles in both physiological and pathological conditions, including GBM pathogenesis, by regulating expression levels of the target genes. Brain cancer stem cells (BCSCs) are subpopulations of brain cancer mass that are responsible for poor prognosis, including therapy resistance and relapse. Epigenetic regulation mediated by miRNAs is also a critical component of BCSC self-renewal and differentiation properties. Propolis is a resinous substance that is collected by honey bees from various plant sources. The flavonoids content of propolis varies, depending on the region collected andthe extraction method. Although the effects of propolis that have been collected from different sources on the miRNA expression levels in the glioblastoma cells have been shown, the effects on the BCSCs are not known yet. OBJECTIVE The aim of this study is to evaluate the effects of Aydin, a city in western Turkey, propolis, on miRNA expression levels of BCSCs and GBM cells. METHODS Aydin propolis was dissolved in 60% ethanol, and after evaporation, distilled water was added to prepare the propolis stock solution. The flavonoids content of the Aydin propolis was determined by MS Q-TOF analysis. Commercially obtained U87MG, GBM cell line, and BCSCs were used as in vitro brain cancer models. The cytotoxic and apoptotic effects of Aydin propolis were determined via WST-1 assay and Annexin V test, respectively. The miRNA expression profile was investigated via the real-time qRT-PCR method, and fold changes were calculated by using the 2-∆∆Ct method compared to untreated control cells. The miRNA-mRNA-pathway interactions, including significantly altered miRNAs, were determined using different bioinformatics tools and databases. RESULTS Quercetin 3-methyl ether was determined as the major component of the Aydin propolis. Aydin propolis did not show significant cytotoxic and apoptotic effects on both GBM and BCSCs up to 2mg/ml concentration. Aydin propolis treatment decreased the expression of nine and five miRNAs in the U87MG 2.13 to 5.65 folds and BCSCs 2.02 to 12.29 folds, respectively. Moreover, 10 miRNAs 2.22 to 10.56 folds were upregulated in propolis treated GBM cells compared to the control group, significantly (p<0.05). In the study, the potential roles of two new miRNAs, whose regulations in glioma were not previously defined, were identified. One of these miR-30d-5p, a novel potential oncomiR in GBM was 2.46 folds downregulated in Aydin propolis treated GBM cells. The other one is miR-335-5p which is a potential tumor suppressor miR in GBM, was 5.66 folds upregulated in Aydin propolis treated GBM cells. FOXO pathway and its upstream and downstream regulators and critically neuronal developmental regulators NOTCH and WNT pathways were determined as the most deregulated pathways in Aydin propolis treated cells. CONCLUSION The determination of the anti-cancer effect of Aydin propolis on the miRNA expression of GBM, especially on cancer stem cells, may contribute to the elucidation of brain cancer genetics by supporting further analyses.