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 Aydın, 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 Aydın 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 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.
Objectives Familial transmission is observed in approximately 10% of cases with type 1 diabetes mellitus (T1DM). The most important gene determining susceptibility is the human leukocyte antigen complex (HLA) located on chromosome 6. More than 50 susceptible loci are associated with T1DM susceptibility have been identified in genes other than HLA. In this study, it was aimed to investigate the molecular genetic etiology by whole-exome sequence (WES) analysis in cases with familial T1DM with no or weakly detected HLA tissue type susceptibility. We aimed to identify new genes responsible for the development of type 1 diabetes and to reveal new genes that have not been shown in the literature before. Methods Cases with at least one T1DM diagnosis in first-degree relatives were included in the study. In the first step, HLA DQ2 and DQ8 loci, which are known to be associated with T1DM susceptibility, were investigated by. In the second step, the presence of variants that could explain the situation was investigated by WES analysis in patients who were negative for both HLA DQ2 and HLA DQ8 haplotypes, HLA DQ2 negative, HLA DQ8 positive, and HLA DQ2 positive and HLA DQ8 negative patients. Results The mean age and duration of diabetes of the 30 cases (Girl/Male: 17/13) were 14.9 ± 6 and 7.56 ± 3.84 years, respectively. There was consanguineous marriage in 5 (16%) of the families. As a result of filtering all exome sequence analysis data of two cases with DQ2 (DQB1*02) (−) and DQ8 (DQB1*03:02) (−), seven cases with DQ2 (DQB1*02) (+) and DQ8 (DQB1*03:02) (−), and one case with DQ2 (DQB1*02) (−) and DQ8 (DQB1*03:02) (+), seven different variants in seven different genes were detected in five cases. The pathogenicity of the detected variants were determined according to the “American College of Medical Genetics and Genomics (ACMG)” criteria. These seven variants detected were evaluated as high-score VUS (Variants of unknown/uncertain significance). In the segregation study conducted for the mutation in the POLG gene detected in case 5, this variant was detected in the mother of the case and his brother with T1DM. Segregation studies are ongoing for variants detected in other affected individuals in the family. Conclusions In conclusion, in this study, seven different variants in seven different genes were detected in five patients by WES analysis in familial T1DM patients with no or weak HLA tissue type susceptibility. These seven variants detected were evaluated as high-score VUS. POLG might be a novel candidate gene responsible for susceptibility to T1DM. Non-HLA genes directly responsible for the development of T1DM were not detected in any of the cases.
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