RAB25沉默抑制结直肠癌细胞铁死亡的作用研究

doi:10.16150/j.1671-2870.2022.06.08

摘要/Abstract

摘要：

目的：探讨沉默RAS相关结合蛋白25（ras-associated binding protein 25, RAB25）在结直肠癌（colorectal cancer，CRC）细胞铁死亡中的作用。方法：利用GEPIA（Gene Expression Profiling Interactive Analysis）数据库分析RAB25的表达水平及与铁死亡关键基因表达之间的关联。在CRC细胞系HCT116上构建慢病毒介导的RAB25沉默细胞株（shRAB25），应用定量实时聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）和蛋白印迹法检测RAB25的表达情况。应用细胞增殖与毒性检测法（CCK8）检测不同浓度的（0~20 μmol/L）铁死亡诱导剂erastin对沉默RAB25后细胞活力的影响；利用荧光显微镜和透射电镜分别观察erastin对沉默RAB25后的细胞形态和线粒体结构的影响；使用C11-BODIPY染色和流式细胞仪检测erastin对沉默RAB25后的细胞脂膜过氧化水平的影响。检测erastin与西妥昔单抗对沉默RAB25后的细胞活力的联合作用。结果：RAB25在CRC中表达升高（P<0.01）；RAB25表达与铁死亡关键基因表达明显相关。当erastin≥10 μmol/L时，与阴性对照组（空载慢病毒感染阴性组）相比，RAB25沉默组抑制了铁死亡导致的细胞杀伤（P<0.000 1），细胞形态和线粒体结构更清晰完整；流式细胞术检测结果提示，细胞脂膜过氧化水平明显下降（P<0.000 1）。RAB25表达使erastin和西妥昔单抗对HCT116细胞的联合杀伤作用明显增强。结果：沉默RAB25可抑制erastin诱导的CRC细胞铁死亡；RAB25可协同铁死亡诱导剂erastin诱导铁死亡，增强西妥昔单抗的疗效。

关键词: 结直肠癌, RAS相关结合蛋白25, 铁死亡

Abstract:

Objective: To investigate the effect of RAB25 on the sensitivity of erastin-induced ferroptosis in colorectal cancer(CRC). Methods: The GEPIA database was used to analyze the mRNA expression levels of RAB25 in CRC tissues and the co-expression between RAB25 and ferroptosis related genes. The lentivirus-mediated RAB25 knockdown cell line was constructed. The expression of RAB25 was detected by quantitative real-time polymerase chain (qRT-PCR) and western blotting. After adding erastin at different concentrations(0-20 μmol/L), the cell proliferation rate was measured by CCK8 assay. The cell morphology and the mitochondrial structure were observed by inverted fluorescence microscope and transmission electron microscope, respectively. C11-BODIPY staining and flow cytometry probe were used to determine the level of lipid peroxides in cellular membranes. The combined effect of erastin and cetuximab on cellular viability in RAB25 knockdown cells were investigated. Results: The mRNA expression of RAB25 was unregulated in colorectal cancer tissues (P<0.01). The mRNA expression of RAB25 was correlated with some vital ferroptosis-associated genes. When erastin ≥10 μmol/L, the shRAB25 group inhibited the killing effect of ferroptosis on cell viability (P<0.000 1) compared to that of the control group, and cell morphology and mitochondrial structure were clearer and complete in the shRAB25 group. Flow cytometry revealed a significant decrease in lipid peroxidation levels in the shRAB25 group(P<0.000 1) compared to the control group. The expression of shRAB25 enhanced the toxic effects by erastin and cetuximab combination. Conclusions: RAB25 is highly expressed in colorectal cancer tissues and played an significant role in ferroptosis. After silencing RAB25, the erastin-induced lethal effect of colorectal cancer cells was inhibited. RAB25 and erastin may induce ferroptosis synergistically, enhancing the efficacy of cetuximab

Key words: Colorectal cancer, Ras-associated binding protein 25, Ferroptosis

中图分类号:

R735.3

李佳曦, 汪锦江, 俞立萍, 袁英, 乔光磊, 马俐君. RAB25沉默抑制结直肠癌细胞铁死亡的作用研究[J]. 诊断学理论与实践, 2022, 21(06): 710-718.

LI Jiaxi, WANG Jinjiang, YU Liping, YUAN Ying, QIAO Guanglei, MA Lijun. Effect of RAB25 knockdown on ferroptosis of colorectal cancer cells[J]. Journal of Diagnostics Concepts & Practice, 2022, 21(06): 710-718.

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