背景:食管鳞状细胞癌(ESCC)仍然是世界范围内最致命的癌症之一,预后极差。因此,本研究旨在筛选影响ESCC的新分子,并探讨其作用机制,为ESCC的靶向治疗提供思路。
方法:首先,我们通过ESCC患者组织的高通量测序筛选出膜蛋白SCARA5,用RT-qPCR和WB验证SCARA5在食管细胞株中的差异表达,和IHC分析SCARA5在ESCC组织中的表达定位。然后,流式细胞术,伤口愈合试验,用Transwell法和CCK-8法研究SCARA5对细胞周期的影响,食管鳞癌细胞的迁移和侵袭以及细胞增殖活性。同时,透射电子显微镜用于检测细胞线粒体形态的变化,和流式细胞术用于检测细胞内活性氧代谢的变化,免疫荧光和流式细胞术检测细胞内Fe2+的变化。机械上,免疫共沉淀用于检测SCARA5是否与铁蛋白轻链结合,WB检测铁凋亡相关蛋白的表达。最后,应用肿瘤异种移植模型验证SCARA5体内肿瘤生长抑制的作用.
结果:我们发现SCARA5在ESCC组织和细胞系中异常降低。此外,我们证实SCARA5抑制了细胞周期,ESCC细胞的转移和侵袭。同时,我们还发现SCARA5的过表达引起线粒体形态的改变,ESCC细胞中细胞内活性氧的积累和细胞内Fe2+的增加,在ESCC细胞中诱导铁凋亡。机械上,我们验证了SCARA5与铁蛋白轻链结合并增加细胞内Fe2。还有,过表达SCARA5通过增加裸鼠皮下肿瘤的铁蛋白轻链来诱导铁凋亡,并抑制裸鼠皮下肿瘤的生长。
结论:总的来说,我们的发现表明,SCARA5通过与铁蛋白轻链结合触发铁凋亡来抑制ESCC的增殖和转移。
BACKGROUND: Esophageal squamous cell carcinoma (ESCC) remains one of the most lethal cancers worldwide accompany with an extremely poor prognosis. Therefore, this study aims to screen for new molecules affecting ESCC and explore their mechanisms of action to provide ideas for targeted therapies for ESCC.
METHODS: Firstly, we screened out the membrane protein SCARA5 by high-throughput sequencing of the ESCC patient tissues, and RT-qPCR and WB were used to verify the differential expression of SCARA5 in esophageal cell lines, and IHC analyzed the expression localization of SCARA5 in ESCC tissue. Then, flow cytometry, wound healing assay, Transwell assay and CCK-8 assay were used to explore the effects of SCARA5 on cell cycle, migration and invasion as well as cell proliferation activity of esophageal squamous carcinoma cells. Meanwhile, transmission electron microscopy was used to detect changes in cellular mitochondrial morphology, and flow cytometry were used to detect changes in intracellular reactive oxygen metabolism, and immunofluorescence and flow cytometry were used to detect changes in intracellular Fe2+. Mechanistically, co-immunoprecipitation was used to detect whether SCARA5 binds to ferritin light chain, and ferroptosis-related protein expression was detected by WB. Finally, the tumor xenograft model was applied to validation the role of SCARA5 tumor growth inhibition in vivo.
RESULTS: We found that SCARA5 was aberrantly decreased in ESCC tissues and cell lines. Furthermore, we confirmed that SCARA5 suppressed the cell cycle, metastasis and invasion of ESCC cells. Meanwhile, we also found that overexpression of SCARA5 caused changes in mitochondrial morphology, accumulation of intracellular reactive oxygen species and increased intracellular Fe2+ in ESCC cells, which induced ferroptosis in ESCC cells. Mechanically, we validated that SCARA5 combined with ferritin light chain and increased intracellular Fe2+. As well as, overexpression SCARA5 induced ferroptosis by increasing ferritin light chain in nude mice subcutaneous tumors and inhibited the growth of nude mice subcutaneous tumors.
CONCLUSIONS: Collectively, our findings demonstrated that SCARA5 suppressed the proliferation and metastasis of ESCC by triggering ferroptosis through combining with ferritin light chain.