Targeting genes within the circRNA-miRNA sponge network using Achyrofuran in a 3D model of SH- SY5Y cells

Aim: In this study, the diagnostic and therapeutic potential of miRNAs and circRNAs in Alzheimer's disease was investigated. The effect of Achyrofuran on circRNA–miRNA interactions targeting Aβ and tau proteins was evaluated, and their roles in gene expression as well as in apoptosis, autophagy, and mitophagy were investigated. Methods: The SH-SY5Y cell line was employed to investigate the molecular mechanisms of Alzheimer's disease associated with Achyrofuran. The cells were differentiated into a neuron-like phenotype through an 18-day protocol involving retinoic acid, BDNF, and db-cAMP. Experimental toxicity and tau models were induced using Aβ oligomers and okadaic acid. Achyrofuran was administered at a dose of 0.07 μg/ml to evaluate its effects. Cell viability and proliferation were assessed using Trypan Blue, CCK-8, and MTT assays, while apoptosis, autophagy, and mitophagy were analyzed with ELISA-based kits. Gene, miRNA, and circRNA expressions were analyzed by qRT-PCR; cell morphology by phase-contrast microscopy; and Tau and Aβ proteins by immunocytochemistry. Data were analyzed using SPSS 11.0, with p<0.05 considered statistically significant. Results: SH-SY5Y cells developed a neuron-like morphology at the end of the 18- day protocol.Achyrofuran treatment reduced cell viability while increasing apoptosis.An increase in BECN1 and LC3-II accompanied by a decrease in p62 indicated autophagy activation.CYCS, CASP3, and CASP9 gene expressions were upregulated, while their targeting miRNAs were suppressed and the corresponding circRNAs were increased.In the mitophagy pathway, PINK1 was upregulated, LRRK2 was downregulated, while no significant change was observed in PARK2.Immunocytochemistry revealed a reduction in Aβ aggregation but persistent mislocalization of tau protein.The findings suggest that Achyrofuran may exert neuroprotective effects through autophagy and mitophagy; however, increased apoptosis and tau pathology limit this effect. Conclusion: This study demonstrated that Achyrofuran modulates autophagy, apoptosis, and mitophagy in SH-SY5Y cells. Although it reduced Aβ accumulation, increased apoptosis and limited mitophagy constrained its effect. The findings suggest that appropriate dosing and combination strategies should be explored in future studies. Keywords: Achyrofuran, Alzheimer's disease, SH-SY5Y cells