摘要
目的探讨circROBO1对视网膜母细胞瘤细胞增殖的影响及其机制。方法体外传代培养视网膜母细胞瘤细胞Y79。取传2代、对数生长期、生长状态良好的Y79细胞,随机分为si-circROBO1组、si-control组,分别转染si-circROBO1、si-control。转染6 h,继续培养24 h,收集细胞,采用RT-qPCR法检测circROBO1表达,采用CCK-8法检测细胞活力,采用细胞克隆形成实验检测细胞增殖能力。通过CircInteractome在线网站预测circROBO1与miR-217的结合位点,并采用双荧光素酶报告基因实验、RNA免疫沉淀实验验证两者的靶向调控关系。结果si-circROBO1组circROBO1相对表达量显著低于si-control组(P<0.05)。si-circROBO1组细胞活力、克隆形成率均显著低于si-control组(P均<0.05)。经CircInteractome在线网站预测,circROBO1序列中可能存在两个与miR-217结合的位点。经双荧光素酶报告基因实验、RNA免疫沉淀实验验证,共转染野生型circROBO1和miR-217可降低Y79细胞荧光素酶活性,并且miR-217主要富集在野生型circROBO1 MS2bs载体转染的Y79细胞中。结论circROBO1可能通过靶向调控miR-217促进视网膜母细胞瘤细胞增殖。
Objective To investigate the effect of circROBO1 on the proliferation of retinoblastoma cells and its mechanism.Methods Retinoblastoma cell line Y79 was subcultured in vitro.Y79 cells in the second passage,which were in logarithmic growth phase and had good growth status,were randomly divided into the si-circROBO1 group and si-control group,which were transfected with si-circROBO1 and si-control,respectively.The cells were transfected for 6 h and continued to be cultured for 24 h.The expression of circROBO1 was detected by RT-qPCR,the cell viability was detected by CCK-8 method,and the cell proliferation was detected by cell cloning experiment.The binding sites of circROBO1 and miR-217 were predicted through the online website of CircInteractome,and the targeted regulatory relationship between the two was verified by double luciferase reporter gene test and RNA immunoprecipitation test.Results The rel⁃ative expression of circROBO1 in the si-circROBO1 group was significantly lower than that in the si-control group(P<0.05).The cell viability and colony-forming ability of the si-circROBO1 group were significantly lower than those of the si-control group(both P<0.05).According to the prediction of CircInteractome online website,there might be two binding sites with miR-217 in circROBO1 sequence.The double luciferase reporter gene test and RNA immunoprecipitation test showed that co-transfection of wild-type circROBO1 and miR-217 could reduce the luciferase activity,and miR-217 was mainly enriched in Y79 cells transfected with wild-type circROBO1 MS2bs vector.Conclusion CircROBO1 may pro⁃mote the proliferation of retinoblastoma cells by targeting miR-217.
作者
刘越峰
张勇
李欣
周霞
罗卫民
LIU Yuefeng;ZHANG Yong;LI Xin;ZHOU Xia;LUO Weimin(Department of Ophthalmology,Taihe Hospital(Affiliated Hospital of Hubei University of Medicine),Shiyan 442000,China;不详)
出处
《山东医药》
CAS
2023年第13期44-47,共4页
Shandong Medical Journal
基金
十堰市太和医院科研项目(2017JJXM119)。
