Hücre Kültüründe Aromataz/Sirtuin-1 Etkileşiminin miRNA İfade Düzeyine Olan Etkisinin Araştırılması

Abstract

Kartal, Y. Evaluation of the Relationship Between Aromatase/Sirtuin-1 Interaction and miRNA Expression in Human Neuroblastoma Cells, Hacettepe University Graduate School of Health Sciences Physiology Programme Doctor of Philosophy Thesis, Ankara 2021. Sirtuin-1 (SIRT1) and aromatase are involved in many physiological and pathological processes and changes in their activation/inhibition play an important role in neurodegenerative diseases. MicroRNAs (miRNAs) are fundamental regulators of gene expression and modulate multiple molecular pathways involved in many diseases. The purpose of this study was to investigate the interaction between aromatase and SIRT1 in human neuroblastoma cells and connection between this interaction and miRNA expressions. In this study, cultured human neuroblastoma cells were incubated in serum-deprived media for 6, 12, 24 h and aromatase and SIRT1 expression were evaluated by Western blot. The IC50 concentration of SIRT1 activator (SRT1720), SIRT1 inhibitor (EX527) and aromatase inhibitors (letrozole and fadrozole) were determined using XTT method. Then, aromatase and SIRT1 levels were evaluated in the presence of SIRT1 siRNA or IC50 values for each of SRT1720, EX527, letrozole and fadrozole. Finally, SIRT1, aromatase expression and miRNA target gene analyzes were assessed with bioinformatics approaches and statistical parameters in all groups. According the results, aromatase and SIRT1 protein levels were significanty elevated in the cells incubated at 24 h in serum-deprived media (p ≤ 0,05). SIRT1 also positively regulated CYP19A1 in SH-SY5Y cells in media with/without FBS. Additionally, SIRT1 siRNA transfection resulted in decreased LC3, ATG5 and LAMP2 expressions in human neuroblastoma cells. In this study, IC50 value as 13.55 μM for SRT1720, 308.22 μM for EX527, 26.81 μM for letrozole and 3 μM for fadrozole in SH-SY5Y with medium phenol red free and in the presence of phenol red, the value of IC50 as 14.45 μM for SRT1720, 316.10 μM for EX527, 23.92 μM for letrozole and 4.57 μM for fadrozole for 24 h were determined. Serum deprivation for 6, 12, 24 and 48 hours caused changes in the oxidant/antioxidant system. Oxidative Stress Index (OSI) tend to decrease in the absence of FBS at 24 h compared to the control and it significantly decreased in the 48 h serum deprived manner (p ≤ 0,001). As a result of bioinformatic analysis, 3 miRNAs which could potentially regulate the SIRT1 and CYP19A1 were determined. As a result of the pathway enrichment analysis using target genes, many pathways related to the nervous system or diseases were detected. In addition, hsa-miR-27a-3p and hsa-miR-181a-5p correlated in terms of their expressions at 24 h compared to 12 h and there was a significant decrease in the expression of these miRNAs. On the other hand, the expression of hsa-miR-30c-5p was significantly increased at 24 h compared to 12 h. These results may provide novel approach for further investigation strategy in terms of complement with existing therapies in future studies and contributed an actual framework in neurology-associated diseases. Keywords: Aromatase, Sirtuin-1, hsa-miR-27a-3p, hsa-miR-30c-5p, hsa-miR-181a-5p, oxidative stress. Supporting Organization: H.U. Scientific Research and Projects Coordination Unit, Project No: THD-2019-18132.