Objective To investigate the regulatory role of PLA2G4A targeting in ferroptosis and its sensitizing effect on the ferroptosis inducer Erastin. Methods PLA2G4A expression in lung adenocarcinoma (LUAD) was assessed by analyzing data from The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium databases, followed by immunohistochemical validation. PLA2G4A expression was knocked down in H1299 lung cancer cells using small interfering RNA. The correlation between PLA2G4A and ferroptosis marker genes was examined through gene correlation analysis and Western blotting. The regulatory relationship between PLA2G4A and ferrous ion (Fe2+) was analyzed using high-content fluorescence imaging. Cell proliferation after PLA2G4A inhibition and Erastin treatment was measured by CCK-8 assay. Flow cytometry and high-content fluorescence imaging were employed to evaluate the effects of PLA2G4A suppression combined with Erastin on intracellular Fe2+ and lipid peroxidation levels. Results Both mRNA (P<0.05) and protein (P<0.001) levels of PLA2G4A were significantly upregulated in LUAD tissues, and its high expression was associated with poor prognosis in LUAD patients (P<0.05). PLA2G4A expression was positively correlated with SLC7A11 expression (r=0.23, P<0.001). PLA2G4A knockdown suppressed SLC7A11 protein expression and increased cellular Fe2+ levels (P<0.01). Compared with the control group, PLA2G4A-silenced cells exhibited significantly reduced viability upon Erastin treatment (P<0.001). Furthermore, Erastin enhanced PLA2G4A targeting-induced Fe2+ accumulation and lipid peroxidation (P<0.001). Conclusion Targeting PLA2G4A induces ferroptosis in lung cancer cells by inhibiting SLC7A11 expression and enhances their sensitivity to Erastin.
ObjectiveTo explore the effects of RAS-selective lethal small molecule 3 (RSL3), Erastin, and tert-butyl hydroperoxide (TBHP) on ferroptosis of rat annulus fibrosus cells (AFCs), and to establish an effective in vitro model of ferroptosis of rat AFCs, providing an experimental basis and theoretical support for the subsequent research on the mechanism of ferroptosis. Methods AFCs were isolated and cultured from the caudal vertebrae of 6-week-old female Sprague-Dawley rats by enzymatic hydrolysis. The optimal treatment concentrations of RSL3, Erastin, and TBHP (used for establishing an in vitro model of ferroptosis in rat AFCs) were screened by the cell counting kit 8 (CCK-8) method. According to the optimal concentrations of each inducer obtained through screening, rat AFCs were divided into control group, RSL3 group, Erastin group, and TBHP group. The expression levels of ferroptosis-related genes [glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), acyl coenzyme A synthase long chain member 4 (ACSL4), solute carrier family 7, member 11 (SLC7A11), and prostaglandin-endoperoxide synthase 2 (PTGS2)], and cell proliferation-related genes [proliferating cell nuclear antigen (PCNA)] were detected by real-time fluorescence quantitative PCR (RT-qPCR), Western blot, and cell immunofluorescence staining; iron (Fe2+) content and malondialdehyde (MDA) content kits were used to evaluate iron accumulation and lipid peroxidation. The levels of intracellular lipid droplets (LDs) and reactive oxygen species (ROS) were determined by cell fluorescence staining to evaluate the level of cellular oxidative stress. Results The optimal concentrations for treating rat AFCs were screened by CCK-8 method and rat AFCs were divided into control group, RSL3 (0.1 μmol/L) group, Erastin (0.5 μmol/L) group, and TBHP (50 μmol/L) group. Both RSL3 group and TBHP group downregulated the mRNA expression levels of GPX4, FTH1, SLC7A11, and PCNA, upregulated the mRNA expression levels of ACSL4 and PTGS2, decreased the protein expression levels of GPX4 and FTH1, and increased the protein expression level of PTGS2 (P<0.05); simultaneously, increased the intracellular Fe2+ and MDA contents, LDs and ROS levels (P<0.05), significantly induced the occurrence of ferroptosis. While the Erastin group upregulated the mRNA expression levels of FTH1, SLC7A11, and PTGS2 (P<0.05); however, it had no significant effect on the mRNA expression levels of GPX4, ACSL4, and PCNA, nor on the protein expression levels of GPX4, FTH1, and PTGS2 (P>0.05), although it increased the intracellular MDA content (P<0.05), but did not significantly change the intracellular Fe2+ content, LDs and ROS levels (P>0.05), failed to induce ferroptosis significantly. Conclusion RSL3 and TBHP are suitable for the establishment of ferroptosis models in rat AFCs, which is helpful for the subsequent study of ferroptosis in intervertebral disc degeneration.