Department of Pediatrics, Faculty of Medicine, Ege University, Izmir, Turkey.
Department of Medical Biology, Faculty of Medicine, Ege University, Izmir, Turkey.
Anticancer Agents Med Chem. 2022;22(2):378-389. doi: 10.2174/1871520621666210504082528.
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.
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.
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 method. The miRNA-mRNA-pathway interactions, including significantly altered miRNAs, were determined using different bioinformatics tools and databases.
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.
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.
IV 级神经胶质瘤被归类为胶质母细胞瘤（GBM），是最恶性的脑癌类型。各种遗传和表观遗传机制在 GBM 的发生和发展中发挥作用。微小 RNA（miRNA）是一种小的非编码 RNA 分子，属于主要的表观遗传调节 RNA 类，在生理或病理条件下发挥不同的作用，包括调节靶基因表达水平的 GBM 发病机制。脑肿瘤干细胞（BCSCs）是导致预后不良的原因，包括治疗耐药和复发。miRNA 介导的表观遗传调控也是 BCSC 自我更新和分化特性的关键组成部分。蜂胶是蜜蜂从各种植物来源中采集的树脂状物质。蜂胶的类黄酮含量因采集区域和提取方法而异。尽管有研究包括不同来源的蜂胶对神经胶质瘤细胞中 miRNA 表达水平的影响，但对 BCSCs 的影响尚未研究。
本研究旨在评估来自土耳其西部城市艾登的蜂胶对 BCSC 和 GBM 细胞 miRNA 表达水平的影响。
将艾登蜂胶溶解在 60%乙醇中，蒸发后加入蒸馏水制备蜂胶储备溶液。通过 MS Q-TOF 分析确定艾登蜂胶的类黄酮含量。使用商业获得的 U87MG 和 BCSCs 作为体外脑癌模型。通过 WST-1 测定法和 Annexin V 试验分别测定 Aydin 蜂胶的细胞毒性和细胞凋亡作用。使用实时 qRT-PCR 方法研究 miRNA 表达谱。通过 2-ΔΔCt 法计算倍数变化。使用不同的生物信息学工具和数据库确定包括显着改变的 miRNA 在内的 miRNA-mRNA-途径相互作用。
艾登蜂胶的主要成分是槲皮素 3-甲醚。艾登蜂胶在高达 2mg/ml 的浓度下对 GBM 和 BCSC 均无明显的细胞毒性和细胞凋亡作用。艾登蜂胶处理降低了 U87MG 中 9 种 miRNA 和 BCSC 中 5 种 miRNA 的表达。此外，与对照组相比，在 Aydin 蜂胶处理的 GBM 细胞中，有 10 种 miRNA 的表达水平显著上调了 2.22 至 10.56 倍（p<0.05）。在研究中，确定了两种新 miRNA 的潜在作用，它们在胶质瘤中的调节作用以前没有定义。其中一种是 miR-30d-5p，它是 GBM 中的一种新型潜在致癌 miRNA，在 Aydin 蜂胶处理的 GBM 细胞中下调了 2.46 倍。另一种是 miR-335-5p，它是 GBM 中的一种潜在肿瘤抑制 miRNA，在 Aydin 蜂胶处理的 GBM 细胞中上调了 5.66 倍。FOXO 途径及其上下游调节剂，以及关键的神经元发育调节剂，NOTCH 和 WNT 途径，被确定为 Aydin 蜂胶处理细胞中最失调的途径。
确定艾登蜂胶对 GBM，特别是癌症干细胞的 miRNA 表达的抗癌作用可能通过支持进一步分析为脑癌遗传学的阐明做出贡献。
