外泌体circRPPH1促进结直肠癌增殖和转移

Exosome circRPPH1 promotes colorectal cancer cell proliferation and metastasis

目的:探讨外泌体circRPPH1在结直肠癌(CRC)细胞中的生物学功能及潜在作用机制。方法:对GEO数据库GSE126094进行分析,筛选并验证CRC组织差异表达circRNA。SW620和SW480细胞转染circRPPH1 shRNA(sh-circRPPH1)后,MTT、流式细胞术和Transwell实验观察细胞增殖活性、细胞周期、迁移和侵袭能力变化。将转染Oe-circRPPH1质粒和sh-circRPPH1的SW620和SW480细胞(供体细胞)分别与未经处理的SW620和SW480细胞(受体细胞)共培养,qRT-PCR检测供体细胞、细胞外泌体及受体细胞circRPPH1表达。在线生物信息学数据和双荧光素酶报告基因实验预测并验证circRPPH1的靶miRNA及其下游靶基因。结果:circRPPH1在CRC肿瘤组织和癌细胞中高表达;敲除circRPPH1可抑制SW620和SW480细胞增殖、阻滞细胞周期、抑制细胞迁移和侵袭。SW620和SW480细胞可通过外泌体将circRPPH1传递给周围癌细胞。生物信息学和荧光素酶报告基因实验显示,circRPPH1在CRC细胞中起miR-330-5p海绵作用,miR-330-5p在CRC肿瘤组织和癌细胞中均呈低表达;NRAS是miR-330-5p的下游靶基因,在CRC肿瘤组织和癌细胞中均呈高表达。结论:circRPPH1在CRC细胞增殖和转移过程中发挥重要作用,并可能通过海绵miRNA发挥调控作用,提示外泌体circRPPH1在CRC中起致癌作用,可能是CRC的潜在生物标志物。

Objective:To investigate biological function of exosome circRPPH1 in colorectal cancer(CRC)cells and its potential mechanism of action.Methods:GEO database GSE126094 was analyzed to screen and verify differentially expressed circRNA in CRC tissues.SW620 and SW480 cells were transfected by circRPPH1 shRNA(sh-circRPPH1),and changes of proliferating activity,cell cycle,migration and invasion ability were observed by MTT,flow cytometry and Transwell assay.SW620 and SW480 cells transfected with Oe-circRPPH1 plasmid and sh-circRPPH1(donor cells)were co-cultured with untreated SW620 and SW480 cells(recipient cells),and expression of circRPPH1 in donor cells,exosomes and recipient cells was detected by qRT-PCR.Targeting miRNAs and their downstream target genes of circRPPH1 were predicted and verified by online bioinformatics data and dual luciferase reporter gene experiments.Results:circRPPH1 was highly expressed in CRC tumor tissues and cancer cells.circRPPH1 knockdown could inhibit proliferation of SW620 and SW480 cells,block cell cycle,and inhibit cell migration and invasion.SW620 and SW480 cells could deliver circRPPH1 to surrounding cancer cells via exosomes.Bioinformatics and luciferase reporter gene experiments showed that circRPPH1 acted as a sponge for miR-330-5p in CRC cells,and miR-330-5p was low expressed in both CRC tumor tissues and cancer cells.NRAS was downstream target gene of miR-330-5p,and highly expressed in CRC tumor tissues and cancer cells.Conclusion:circRPPH1 plays an important role in proliferation and metastasis of CRC cells,and may play regulatory role through sponge miRNA,suggesting that exosome circRPPH1 plays a carcinogenic role in CRC and may be a potential biomarker of CRC.