摘要
Abstract
Objective To investigate the role and mechanisms of acyl-CoA synthetase long-chain family member 3(ACSL3)m6A modification in suppressing ferroptosis during cisplatin resistance in non-small cell lung cancer.Methods In vitro,cisplatin-resistant human lung adenocarcinoma A549/DDP cells were subjected to the following interventions:transduction with lentiviral vectors expressing ACSL3 short hairpin RNA,transfection with Wilms'tumor 1-associating protein(WTAP)small interfering RNA(siRNA)plasmids,or co-transfection with WTAP siRNA plasmids and ACSL3 overexpression plasmids.After 48 hours,all the groups were replaced with complete medium containing 1 μg/mL cisplatin and continued to culture for 24 h.A549(or A549/DDP)cells infected with ACSL3 short hairpin RNA lentivirus were subcutaneously inoculated into the backs of nude mice to establish a subcutaneous xenograft model.Immunofluorescence assay was performed to detect ACSL3 protein expression in cisplatin-treated or normally cultured A549 and A549/DDP cells.Cisplatin sensitivity was evaluated using colony formation assays.FerroOrange staining and total iron assay kits were used to measure intracellular and tissue iron levels.Lipid peroxidation was detected using the BODIPY 581/591 C11 probe.ACSL3 m6A methylation was analyzed by MeRIP-qPCR,while RNA decay assays evaluated ACSL3 mRNA stability.qRT-PCR and Western blotting were used to measure mRNA and protein expressions of ACSL3 and WTAP.Ferritin and COX2 levels were measured by immunohistochemistry.Results After cisplatin treatment,the decrease in ACSL3 expression was significantly smaller in A549/DDP cells[(48.70±3.81)％]than in A549 cells[(65.62±2.53)％].ACSL3 knockdown significantly reduced the colony formation capacity of A549/DDP cells,but increased FerroOrange intensity,total iron levels and lipid peroxidation levels(P＜0.05).Cisplatin reduced ACSL3 m6A methylation in A549/DDP cells,with a less decline than in A549 cells(P＜0.05).WTAP knockdown accelerated ACSL3 mRNA decay and reduced m6A enrichment of ACSL3.Compared to A549/DDP cells with only WTAP knockdown,those with combined si-WTAP knockdown and ACSL3 overexpression showed increased colony formation capacity,but decreased FerroOrange intensity,total iron levels,and lipid peroxidation levels.In vivo,ACSL3 knockdown upregulated COX2 and ferritin levels,increased Fe2+and ROS levels in tumor tissues,and suppressed tumor growth.Conclusion WTAP suppresses ferroptosis and reduces cisplatin sensitivity in A549 cells by mediating m6A modification of ACSL3 mRNA,thereby regulating the progression of lung cancer.关键词
肺癌/铁死亡/顺铂耐药/酰基辅酶A合成酶长链家族成员3(ACSL3)/Wilms肿瘤1相关蛋白(WTAP)Key words
lung cancer/ferroptosis/cisplatin resistance/acyl-CoA synthetase long-chain family member 3(ACSL3)/Wilms' tumor 1-associating protein(WTAP)分类
医药卫生
