背景:由于EIF3D在结直肠癌(CRC)中是致癌的,并且与多药耐药有关,本研究旨在探讨EIF3D是否以及如何调节CRC对5-氟尿嘧啶(5-Fu)的耐药性。
方法:使用生物信息学工具预测CRC中EIF3D相关基因。CRC细胞和裸小鼠接受5-Fu处理。然后,EIF3D以及EIF3D和RUVBL1之间的相互作用对细胞活力的影响,菌落形成,凋亡,通过MTT检测DNA损伤,菌落形成,流式细胞术,和免疫荧光分析,以及通过鼠异种移植试验对体内肿瘤发生的研究。通过probit回归分析确定5-Fu对CRC细胞的IC50值。EIF3D的表达式,eIF4E,EIF3D相关基因,γH2AX,Bcl-2,Bax,以及在CRC组织中裂解的Caspase-3/Caspase-3,细胞,和/或通过qRT-PCR和/或Western印迹分析异种移植肿瘤。
结果:EIF3D和RUVBL1高表达,与CRC组织/细胞呈正相关。在CRC细胞中,除了eIF4E,EIF3D和RUVBL1水平均被5-Fu处理上调;除此之外,RUVBL1水平被EIF3D沉默而不是eIF4E下调。同时,EIF3D沉默降低了5-Fu的IC50值,并增强了5-Fu诱导的生存力降低,集落形成抑制,凋亡促进,Bcl-2下调,和γH2AX,Bax,和裂解的Caspase-3/Caspase-3上调,但逆转了5-Fu触发的RUVBL1上调。RUVBL1过表达抵消了EIF3D沉默诱导的5-Fu处理的CRC细胞的活力降低和凋亡促进,以及5-Fu处理小鼠的肿瘤发生抑制和凋亡促进。
结论:EIF3D通过上调RUVBL1水平促进CRC对5-Fu的耐药性。
BACKGROUND: As EIF3D is oncogenic in colorectal cancer (CRC) and is associated with multidrug resistance, this study aims to investigate whether and how EIF3D regulates resistance to 5-fluorouracil (5-Fu) in CRC.
METHODS: EIF3D-associated genes in CRC were predicted using bioinformatics tools. CRC cells and nude mice received 5-Fu treatment. Then, the impacts of EIF3D and the interaction between EIF3D and
RUVBL1 on cell viability, colony formation, apoptosis, and DNA damage were detected through MTT, colony formation, flow cytometry, and immunofluorescence assays, and those on in vivo tumorigenesis through murine xenograft assay. IC50 value of 5-Fu for CRC cells was determined by probit regression analysis. Expressions of EIF3D, eIF4E, EIF3D-associated genes, γH2AX, Bcl-2, Bax, and Cleaved Caspase-3/Caspase-3 in CRC tissues, cells, and/or xenograft tumors were analyzed by qRT-PCR and/or Western blot.
RESULTS: EIF3D and
RUVBL1 were highly expressed and positively correlated with CRC tissues/cells. In CRC cells, except for eIF4E, both EIF3D and RUVBL1 levels were upregulated by 5-Fu treatment; in addition to that,
RUVBL1 level was downregulated by EIF3D silencing rather than eIF4E. Meanwhile, EIF3D silencing diminished IC50 value of 5-Fu and potentiated 5-Fu-induced viability decrease, colony formation inhibition, apoptosis promotion, Bcl-2 downregulation, and γH2AX, Bax, and Cleaved Caspase-3/Caspase-3 upregulation but reversed 5-Fu-triggered RUVBL1 upregulation.
RUVBL1 overexpression offsets EIF3D silencing-induced viability decrease and apoptosis promotion of 5-Fu-treated CRC cells, and tumorigenesis suppression and apoptosis promotion in 5-Fu-treated mice.
CONCLUSIONS: EIF3D promotes resistance to 5-Fu in CRC through upregulating
RUVBL1 level.