METHODS: We first evaluated the expression level of circ-RAPGEF5 in CRC tissues and cells by quantitative real-time polymerase chain reaction (qRT-PCR). Then, we analyzed cell proliferation (EdU and colony formation assay), migration (cell wound healing assay), invasion (transwell assay), and apoptosis (flow cytometry assay). To further elucidate the mechanism of circ-RAPGEF5 in CRC, bioinformatics tools, Dual-luciferase reporter assay, Ago2 RNA immunoprecipitation assay, and RNA pull-down assay were employed. Moreover, we established a CRC transplantation tumor model to evaluate the effect of circ-RAPGEF5 on tumor growth in vivo.
RESULTS: circ-RAPGEF5 was significantly upregulated in CRC tissues and CRC cells. Furthermore, the downregulation of circ-RAPGEF5 restrained CRC cell proliferation, migration, and invasion, and promoted cell apoptosis in vitro. Mechanistically, circ-RAPGEF5 accelerated the malignant behaviors of CRC cells by sponging miR-545-5p, which targeted polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). In addition, we revealed that circ-RAPGEF5 silence curbed tumor growth in vivo.
CONCLUSIONS: These findings revealed that circ-RAPGEF5 played an oncogenic role through the miR-545-5p/GALNT3 axis in CRC progression, providing potential therapeutic targets for the treatment of CRC.
方法:我们首先通过定量实时聚合酶链反应(qRT-PCR)评估了circ-RAPGEF5在CRC组织和细胞中的表达水平。然后,我们分析了细胞增殖(EdU和集落形成测定),迁移(细胞伤口愈合试验),入侵(transwell分析),和细胞凋亡(流式细胞术测定)。为了进一步阐明circ-RAPGEF5在CRC中的作用机制,生物信息学工具,双荧光素酶报告基因测定,Ago2RNA免疫沉淀测定,和RNA下拉分析。此外,我们建立了CRC移植瘤模型来评估circ-RAPGEF5对体内肿瘤生长的影响。
结果:circ-RAPGEF5在CRC组织和CRC细胞中显著上调。此外,circ-RAPGEF5下调抑制CRC细胞增殖,迁移,和入侵,促进细胞凋亡。机械上,circ-RAPGEF5通过增强miR-545-5p加速CRC细胞的恶性行为,其靶向多肽N-乙酰半乳糖胺转移酶3(GALNT3)。此外,我们发现circ-RAPGEF5沉默抑制了体内肿瘤的生长。
结论:这些发现揭示了circ-RAPGEF5通过miR-545-5p/GALNT3轴在CRC进展中发挥致癌作用,为CRC的治疗提供潜在的治疗靶点。