circ_ATP5A1 sponges hsa-miR-145-5p to regulate ZFP36 and ferroptosis-associated signaling in acute kidney injury

Abstract

Circular RNAs (circRNAs) are a novel class of non-coding RNAs that function as competing endogenous RNAs (ceRNAs) by sharing microRNA (miRNA) response elements with mRNAs, thereby modulating gene expression in various biological processes, including ferroptosis. Accumulating evidence has implicated circRNAs in the progression of acute kidney injury (AKI); however, whether circRNAs are involved in the regulation of ferroptosis during AKI and the underlying molecular mechanisms remain unclear. A circRNA-miRNA-mRNA interaction network was initially built via bioinformatics. Quantitative real-time PCR (RT-qPCR), Western blot, and immunohistochemistry were used to measure circ_ATP5A1, hsa-miR-145-5p, and ZFP36 expression. Dual-luciferase reporter assays combined with RNA immunoprecipitation verified the targeting relationships among the three molecules. In LPS-treated HK-2 cells, cell viability, inflammatory responses, and ferroptosis were evaluated using CCK-8, lactate dehydrogenase (LDH) release assay, flow cytometry, ELISA, and commercial reagent kits. An LPS-induced AKI mouse model was also established to assess the protective effect of circ_ATP5A1 expression. A ceRNA regulatory network comprising circ_ATP5A1/hsa-miR-145-5p/ZFP36 was established through bioinformatics prediction and experimental validation. Experimental findings revealed that expression of circ_ATP5A1 and ZFP36 was downregulated in both LPS-challenged HK-2 cells and AKI mice, whereas hsa-miR-145-5p was upregulated. Mechanistic studies demonstrated that circ_ATP5A1 acts as a ceRNA by sponging hsa-miR-145-5p, thereby relieving the repressive effect of hsa-miR-145-5p on its downstream target ZFP36. Functional assays showed that overexpression of circ_ATP5A1 restored the viability of LPS-exposed HK-2 cells, reduced inflammatory cytokine secretion (IL-1β, IL-6, TNF-α), increased the expression of ferroptosis-related markers glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11), and decreased acyl-CoA synthetase long-chain family member 4 (ACSL4) expression. In vivo, circ_ATP5A1 overexpression significantly ameliorated renal dysfunction and tissue injury in AKI mice. The circ_ATP5A1/hsa-miR-145-5p/ZFP36 axis attenuates AKI by inhibiting ferroptosis, providing a theoretical basis for AKI treatment.