circROBO1对视网膜母细胞瘤细胞增殖的影响及其机制

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。

circROBO1上调KLF5促进视网膜细胞瘤侵袭的机制研究

目的:探索circROBO1通过上调KLF5促进视网膜Y79细胞侵袭的作用及其可能的调控机制。方法:RNase R酶消化及qRT-PCR实验检测环状RNA circROBO1在视网膜Y79细胞中的结构稳定性;分别提取视网膜Y79细胞的胞质及胞核RNA,进行circROBO1的亚细胞定位分析;采用siRNA沉默视网膜Y79细胞中circROBO1的表达,划痕实验、Transwell细胞侵袭与迁移实验检测circROBO1对Y79细胞迁移与侵袭能力的影响;通过CircInteractome及TargetScan在线软件分别预测circROBO1与下游miRNA及miRNA与下游靶基因KLF5的靶向结合位点,并采用双荧光素酶报告基因实验验证两者之间的靶向调控关系。蛋白免疫印迹实验检测siRNA沉默Y79细胞中circROBO1的表达后对KLF5表达的影响。结果:与未经RNase R酶处理的对照组相比,circROBO1的相对表达量在RNase R酶处理后并无显著变化;而线性ROBO1的相对表达量在经RNase R酶处理后,则显著降低(t=16.18,P<0.05);circROBO1在细胞质中的含量显著高于细胞核(t=13.04, P<0.05);与转染si-control的对照组相比,si-circROBO1组视网膜Y79细胞的迁移率、Transwell细胞侵袭与迁移能力均显著降低(t=22.54,P<0.05);circROBO1可靶向吸附miR-885-5p、miR-885-5p与KLF5之间存在靶向结合位点(t=11.39,P<0.05);与si-control组相比,si-circROBO1组的KLF5蛋白表达显著降低(t=17.26,P<0.05)。结论:circROBO1通过上调KLF5促进视网膜Y79细胞瘤侵袭。

沉默circROBO1通过下调KRAS抑制鼻咽癌CNE2细胞侵袭与肺转移

目的 探讨沉默环状RNA hsa_circ_0124696(circROBO1)对鼻咽癌CNE2细胞侵袭与肺转移的影响机制。方法 qRT-PCR检测鼻咽癌及癌旁组织中circROBO1表达。采用干扰小RNA(siRNA)沉默鼻咽癌CNE2细胞中circROBO1的表达,Transwell及HE染色检测circROBO1对CNE2细胞迁移能力和体内肺转移的影响。TargetScan在线软件预测circROBO1下游miR-217与下游靶基因KRAS的靶向结合位点,双荧光素酶报告基因实验验证两者之间的靶向调控关系。蛋白免疫印迹检测siRNA沉默CNE2细胞中circROBO1表达对KRAS的影响。结果 鼻咽癌组织中circROBO1表达高于癌旁组织(P<0.05)。与转染si-circNC的对照组相比,si-circROBO1组鼻咽癌CNE2细胞侵袭与体内肺转移能力均显著降低(P<0.05)。circROBO1下游miR-217与KRAS之间存在靶向结合位点,并且circROBO1可影响KRAS的蛋白和mRNA表达量。结论 沉默circROBO1通过miR-217下调KRAS抑制鼻咽癌CNE2细胞侵袭与肺转移。

CircROBO1 promotes retinal Y79 cell tumor invasion by targeting KLF5

Objective:To explore the role of circROBO1 in promoting the invasion of retinal Y79 cells by targeting KLF5 and its possible regulatory mechanism.Methods:RNase R enzyme digestion and qRT-PCR experiments were used to detect the structural stability of circular circROBO1 in retinal Y79 cells;cytoplasmic and nuclear RNAs of retinal Y79 cells were extracted for localization analysis of circROBO1;The expression of circROBO1 in retinal Y79 cells were silenced by siRNA.The effect of circROBO1 on the migration and invasion ability of HT-29 cells was detected by scratch assay,Transwell cell invasion and migration assay.The target binding sites of circROBO1 and its downstream miRNA and that of miRNA and its downstream target gene KLF5 were predicted by CircInteractome and TargetScan online software respectively,and the target regulation relationship between them was verified by double luciferase reporter gene experiment.Western blot was used to detect the effect of siRNA silencing the expression of circROBO1 in Y79 cells on the expression of KLF5.Results:Compared with the control group without RNase R enzyme treatment,relative circROBO1 levels did not change significantly after treatment,while relative linear ROBO1 levels decreased significantly after treatment(t=16.18,P<0.05);the content of circROBO1 in the cytoplasm was significantly higher than that in the nucleus(P<0.05);compared with si-control group,the migration rate and the invasion and migration abilities of Transwell cells were all lower in the si-circROBO1 group(t=22.54,P<0.05);circROBO1 can adsorb miR-885-5p,and there is a target binding site between miR-885-5p and KLF5(t=11.39,P<0.05);compared with the si-control group,the KLF5 protein expression in the si-circROBO1 group was significantly decreased(t=17.26,P<0.05).Conclusions:circROBO1 promotes retinalY79 cell tumor invasion by targeting KLF5.