Propolis Extract Regulates microRNA Expression in Glioblastoma and Brain Cancer
Stem Cells
Authors: C.Yilmaz Ugur , Bagca Bakiye Goker , Karaca Emin , Durmaz Asude , Durmaz Burak , Aykut Ayca , Kayalar Husniye , Avci Cigir Biray , Susluer Sunde Yilmaz , Pariltay Erhan , Gunduz Cumhur , Cogulu Ozgur 
Published: May 2021

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 belong to the main epigenetic regulatory RNA class
that plays different roles in either physiological or pathological conditions, including GBM pathogenesis regulating
expression levels of the target genes. Brain Cancer Stem Cells (BCSCs) are responsible for poor prognosis, including
therapy resistance and relapse. Epigenetic regulation mediated by miRNAs is also a critical component of BCSC selfrenewal
and differentiation properties. Propolis is a resinous substance collected by honey bees from various plant
sources. The flavonoid content of propolis varies depending on the collection region and the extraction method. Although
there are studies that include the effects of different originated-propolis on the miRNA expression levels of the
glioblastoma cells, the impact on the BCSCs has not been studied yet.

Objective:
This study aims to evaluate the effects of propolis obtained from Aydın, a city in western Turkey, 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 and BCSCs were used as in-vitro brain cancer models. Cytotoxic and apoptotic effects of
Aydın propolis were determined via WST-1 assay and Annexin V test, respectively. The miRNA expression profile
was investigated using the real-time qRT-PCR method. The fold changes were calculated by the2-ΔΔCt method. The
miRNA-mRNA-pathway interactions, including significantly altered miRNAs, were determined using different bioinformatics
tools and databases.

Results:
Quercetin 3-methyl ether was the main 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 miRNAs in the U87MG and five miRNAs in the BCSCs. Moreover, ten miRNAs
have upregulated from 2.22 to 10.56 folds 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 them was miR-30d-5p, a novel potential oncomiR in GBM, which 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, that was 5.66 folds upregulated in Aydin propolis treated GBM cells. FOXO pathway, 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 Aydın 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.