重组新城疫病毒rL-RVG通过p53-YAP1-ACSL4通路诱导肺腺癌细胞铁死亡

Recombinant Newcastle disease virus rL-RVG induces ferroptosis in lung adenocarcinoma cells through the p53 YAP1-ACSL4 pathway

目的旨在探究重组新城疫病毒rL-RVG是否通过p53-YAP1-ACSL4通路影响肺腺癌细胞铁死亡。方法体外培养人肺腺癌细胞系A549、PC9,将处于对数增殖期的细胞分为对照组(NC组)、新城疫病毒感染组(NDV组)、重组新城疫病毒感染组(rL-RVG组)、铁死亡诱导剂组(Erastin组)及铁死亡抑制剂组(NAC组)。在病毒感染和铁死亡诱导剂、抑制剂干预后,通过CCK-8、划痕实验和Transwell来检测细胞的功能学变化,包括细胞活力、迁移能力和侵袭能力;光学显微镜观察细胞形态改变。使用流式细胞术和荧光显微镜来测定细胞中ROS的含量,用酶标仪对MDA含量进行测定,通过Western blot和实时荧光定量PCR来检测铁死亡相关蛋白p53、YAP1及ACSL4的表达。结果与NC组相比,rL-RVG组细胞生长、迁移及侵袭能力明显下降(P<0.01);ROS及MDA水平升高且与铁死亡诱导剂组相比含量显著提高(P<0.01),铁死亡抑制剂干预后含量均减少(P<0.01);铁死亡关键蛋白p53、YAP1、ACSL4表达量明显升高(P<0.01);Si-RNA敲减YAP1和ACSL4后相应蛋白的表达量减少(P<0.01),并且ROS和MDA含量均减少(P<0.01)。结论rL-RVG可以有效地阻止肺腺癌细胞的增殖、迁移和扩散,并且能够通过p53-YAP1-ACSL4轴增加脂质过氧化物和细胞活性氧的含量,最终诱导肿瘤细胞铁死亡。

Objective The purpose of this study is to explore whether the recombinant Newcastle disease virus rL-RVG affects ferroptosis in lung adenocarcinoma cells through the p53 YAP1-ACSL4 pathway.Methods A549 and PC9 cell lines were cultured in vitro and cells in logarithmic proliferation were divided into control group(NC),Newcastle disease virus infection group(NDV),recombinant Newcastle disease virus infection group(rL-RVG),ferroptosis inducer group(Erastin)and ferroptosis inhibitor group(NAC).After viral infection and intervention with ferroptosis inducer and inhibitor,we determined the functional changes of cells,including cell vitality,migration ability,and invasion ability,using CCK-8,scratch experiments,and Transwell,and observed morphological changes in cells through optical microscopy.In addition,we used flow cytometry and fluorescence microscopy to determine the content of ROS in cells,and used enzyme-linked immunosorbent assay to measure MDA content,and used Western blot and real-time PCR to detect the expression of ferroptosis related proteins p53,YAP1,and ACSL4.Results Compared to NC group,a significant decrease of cell growth,migration,and invasion of rL-RVG group was observed(P<0.01).ROS and MDA levels were significantly augmented(P<0.01).The levels of ROS and MDA increased significantly compared to the ferroptosis inducer group(P<0.01),while the content of ferroptosis inhibitor decreased after intervention(P<0.01).The expression of ferroptosis key proteins p53,YAP1,and ACSL4 were significantly augmented(P<0.01).After knocking down YAP1 and ACSL4 by Si-RNA,the level of corresponding proteins were decreased(P<0.01),and the content of ROS and MDA were also decreased(P<0.01).Conclusion rL-RVG can effectively prevent the proliferation,migration,and diffusion of lung adenocarcinoma cells,and increase the content of lipid peroxides and cellular reactive oxygen species through the p53 YAP1-ACSL4 axis,ultimately promoting ferroptosis of tumor cells.