Department of Urology, The Fifth Affiliated Hospital, Southern Medical University, Guangzhou, China.
Department of Pathology, Nanfang Hospital and School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.
Mol Carcinog. 2023 Jul;62(7):907-919. doi: 10.1002/mc.23533. Epub 2023 May 8.
Although Poly C Binding Protein 1 (PCBP1) affects cellular ferroptosis and mitochondrial dysfunction, the mechanisms by which PCBP1 regulates bladder cancer (BC) cell functions are unknown. In this study, two BC cell lines (T24 and UMUC3) were treated with different doses of ferroptosis inducer erastin to analyze the effect of PCBP1. Online databases (RPISeq and CatRAPID) were used to predict the possible direct interaction between PCBP1 protein and serine β-lactamase-like protein (LACTB) mRNA, which was further validated via RNA pull-down, RNA immunoprecipitation, and luciferase reporter assays. Mitochondria injury and ferroptosis were evaluated using CCK-8 assay, TUNEL staining, flow cytometry, corresponding kits, and JC-1 staining. In vivo experiments were conducted using tumor xenograft models. Quantitative reverse-transcription polymerase chain reaction was used to detect transcript expression levels, while protein levels were analyzed using western blot and immunohistochemistry. PCBP1 expression was significantly upregulated in BC tissues and cell lines. Also, PCBP1 knockdown increased erastin-mediated ferroptosis in T24 and UMUC3 cells, while PCBP1 overexpression decreased erastin-mediated ferroptosis in T24 and UMUC3 cells. Mechanistic results showed that LACTB mRNA is a novel PCBP1-binding transcript. LACTB upregulation promoted erastin-induced ferroptosis and mitochondrial dysfunction. Furthermore, LACTB overexpression reversed PCBP1-mediated ferroptosis protection, including decreased ROS and enhanced mitochondrial function, which were further alleviated after phosphatidylserine decarboxylase (PISD) overexpression. Moreover, PCBP1 silencing significantly enhanced tumor inhibition effect of sulfasalazine in xenograft mice transplanted with T24 and UMUC3 cells, leading to LACTB upregulation and PISD downregulation. In conclusion, PCBP1 protects BC cells against mitochondria injury and ferroptosis via LACTB/PISD axis.
尽管多聚 C 结合蛋白 1（PCBP1）影响细胞铁死亡和线粒体功能障碍，但 PCBP1 调节膀胱癌（BC）细胞功能的机制尚不清楚。在这项研究中，用不同剂量的铁死亡诱导剂 erastin 处理两种 BC 细胞系（T24 和 UMUC3），以分析 PCBP1 的作用。在线数据库（RPISeq 和 CatRAPID）用于预测 PCBP1 蛋白与丝氨酸β-内酰胺酶样蛋白（LACTB）mRNA 之间可能的直接相互作用，进一步通过 RNA 下拉、RNA 免疫沉淀和荧光素酶报告基因测定进行验证。使用 CCK-8 测定、TUNEL 染色、流式细胞术、相应试剂盒和 JC-1 染色评估线粒体损伤和铁死亡。使用肿瘤异种移植模型进行体内实验。定量逆转录聚合酶链反应用于检测转录表达水平，而蛋白质水平则使用 Western blot 和免疫组化进行分析。PCBP1 在 BC 组织和细胞系中表达明显上调。此外，PCBP1 敲低增加了 T24 和 UMUC3 细胞中 erastin 介导的铁死亡，而 PCBP1 过表达则降低了 T24 和 UMUC3 细胞中 erastin 介导的铁死亡。机制研究结果表明，LACTB mRNA 是一种新型的 PCBP1 结合转录本。LACTB 的上调促进了 erastin 诱导的铁死亡和线粒体功能障碍。此外，LACTB 的过表达逆转了 PCBP1 介导的铁死亡保护作用，包括降低 ROS 和增强线粒体功能，而过表达磷脂酰丝氨酸脱羧酶（PISD）后进一步缓解。此外，PCBP1 沉默显著增强了磺胺嘧啶在移植 T24 和 UMUC3 细胞的异种小鼠中的肿瘤抑制作用，导致 LACTB 上调和 PISD 下调。总之，PCBP1 通过 LACTB/PISD 轴保护 BC 细胞免受线粒体损伤和铁死亡。
