METTL14介导ACSL4的m6A甲基化在髓核细胞铁死亡和细胞衰老中的作用

Role of METTL14-mediated m6A methylation of ACSL4 in ferroptosis and senescence of nucleus pulposus cells

目的探究METTL14介导ACSL4的m6A甲基化在髓核细胞铁死亡和细胞衰老中的作用。方法采用市售人髓核细胞,利用叔丁基氢过氧化物复制细胞模型,分别复制METTL14过表达和敲除的稳转细胞株。通过分光光度法检测细胞内亚铁离子、MDA、GSH、GPX4水平,流式细胞术检测ROS和细胞线粒体膜电位,采用β-半乳糖苷酶染色评估细胞衰老情况。同时进行了甲基化RNA免疫共沉淀结合实时荧光定量聚合酶链反应(MeRIP-qRT-PCR)和RNA稳定性实验,探究METTL14对ACSL4表达的调控作用。结果METTL14过表达在髓核细胞中导致显著的铁过载(P<0.05)、ROS相对表达量升高(P<0.05)、MDA水平增加(P<0.05),以及GSH与GPX4水平下降(P<0.05),同时降低线粒体膜电位(P<0.05),从而促进了细胞衰老。通过MeRIP-qRT-PCR和RNA稳定性实验发现METTL14调控ACSL4的表达,并影响其mRNA的稳定性。挽救实验进一步证实METTL14通过调控ACSL4促进髓核细胞衰老的机制。结论METTL14通过介导ACSL4的m6A甲基化过程促进髓核细胞的铁死亡和衰老,为深入理解髓核细胞相关疾病的发病机制提供新的视角和治疗靶点。

Objective To elucidate the role of METTL14-mediated m6A methylation of ACSL4 in ferroptosis and senescence of nucleus pulposus cells.Methods Human nucleus pulposus cells were utilized to establish a tert-butyl hydroperoxide-induced cell model.Stable cell lines were generated with either overexpression or knockdown of METTL14.Intracellular levels of ferrous ions,MDA,GSH,and GPX4 were quantified using spectrophotometry,while ROS and mitochondrial membrane potential were measured via flow cytometry.Cell senescence was evaluated throughβ-galactosidase staining.The methylated RNA immunoprecipitation(MeRIP)and quantitative real-time polymerase chain reaction(qRT-PCR),as well as RNA stability experiments,were performed to investigate the regulatory effects of METTL14 on ACSL4 expression.Results METTL14 overexpression in nucleus pulposus cells caused significant iron overload(P<0.05),increased ROS levels(P<0.05),elevated MDA levels(P<0.05),a significant decrease in GSH and GPX 4 levels(P<0.05),and a reduction in the mitochondrial membrane potential(P<0.05),thus promoting cellular senescence.Using MeRIP-qRT-PCR and RNA stability assays,we found that METTL14 regulated the expression of ACSL4 and affected its mRNA stability(P<0.05).The rescue experiment further confirmed that METTL14 promoted nucleus pulposus cell senescence by regulating ACSL 4(P<0.05).Conclusions This study unveils the role of METTL14 in promoting ferroptosis and senescence of nucleus pulposus cells via mediating m6A methylation of ACSL4,offering a novel perspective and potential therapeutic target for understanding the pathogenesis of diseases involving nucleus pulposus cells.