Abstract:

Objective To explore the role of circ_0001126 in homocysteine （Hcy）-induced ferroptosis of Mouse Podocyte Cell line-5 （MPC-5） cells. Methods MPC-5 cells were cultured in vitro and divided into a control group （0 μmol/L Hcy） and an Hcy group （80 μmol/L Hcy）. After 48 hours of cell intervention， the expressions of glutathione peroxidase 4 （GPX4） and solute carrier family 7 member 11 （SLC7A11） proteins were detected by Western blot. The levels of malondialdehyde （MDA） and glutathione （GSH） were measured using a kit， and the level of Fe2? was observed using a fluorescence assay kit. High-throughput sequencing was employed to screen specific circular RNA （circRNA） in podocytes from the control group and the Hcy group， and quantitative reverse transcription polymerase chain reaction （qRT-PCR） was used for validation. Bioinformatics was used to predict chromosome location and conservation. After transfection with si-circ_0001126 and its negative control （si-NC）， the expressions of GPX4 and SLC7A11 proteins and their mRNAs were detected by Western blot and qRT-PCR， respectively. The intracellular MDA level was measured by a malondialdehyde assay kit， the intracellular GSH level was measured by a GSH assay kit， and the intracellular Fe2? level was observed by a fluorescence assay kit. Results Compared with the Control group， the Hcy group exhibited a significant decrease in the expression of ferroptosis related proteins GPX4 and SLC7A11（P < 0.001）， a notable increase in the expression levels of Fe²⁺ and MDA（P < 0.001）， and a marked decrease in GSH levels（P < 0.001）； High throughput sequencing revealed a total of 12 circRNAs with differential expression in the Hcy group， including 8 up-regulated and 4 down-regulated. Based on |log₂foldchange| （≥ 2） and P value （P < 0.05）， circ_0001126 was screened out， and qRT-PCR was used to verify its upregulation in the Hcy group， which is consistent with the sequencing results（P < 0.001）. Analysis using the UCSC Genome Browser Gateway and circbase showed that circ_0001126 is primarily located at chr3：51659420-51660998， formed by the cyclization of the second exon of the Maml3 gene and is highly conserved. Transfection of si-circRNAs demonstrated that si-circ_0001126-309 had the optimal interference efficiency （P < 0.001）. Compared with the Hcy + si-NC group， the Hcy + si-circ_0001126 group showed a significant increase in the expression of ferroptosis-related proteins GPX4 and SLC7A11 （P < 0.001）， a notable decrease in Fe²⁺ and MDA expression levels（P < 0.001）， and an marked increase in GSH levels（P < 0.001）. Conclusion circ_0001126 shows a significant up-regulation in MPC-5 cells treated with Hcy， and down-regulating its expression can inhibit Hcy-induced ferroptosis in MPC-5 cells.

Key words: circ_0001126, homocysteine, chronic kidney disease, podocytes, ferroptosis