miR-216b在体外培养细胞中加速长非编码RNA UCA1的降解  

miR-216b Accelerated Decay of lncRNA-UCA1 in Cultured Cells

作　　者：郑伟[1] 郭晓华[1] 董洁[1] 李少华[1] 丁红梅[1] 李慧[1] 黄皑雪[1] 夏伟[1] 白琛俊 李达[1] 耿介[1] 李洁[1] 邵宁生[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850

出　　处：《生物技术通讯》2017年第3期227-232,共6页Letters in Biotechnology

基　　金：国家自然科学基金(31370794;31570817;31200566)

摘　　要：目的:探讨长非编码RNA(lncRNA)UCA1作为miR216b的"分子海绵"结合miR-216b后的命运变化。方法:提取人HEK293T细胞基因组,以特异性引物PCR扩增UCA1基因并克隆至pcDNA6.0b载体,用氨苄西林抗性筛选阳性克隆,重组质粒pcDNA6-UCA1转染HEK293T和HeLa细胞并鉴定其表达水平。向HEK293T和HeLa细胞转染miR-216b类似物,同时用放线菌素D抑制细胞转录,收取3个时间点的细胞总RNA并鉴定其完整性,反转录获得cDNA后,采用实时荧光定量PCR技术检测各时间点UCA1的水平,测定其半衰期。pcDNA6-UCA1转染细胞24h后,转染miR-216b类似物或miR-216b抑制剂,并使用放线菌素D抑制转录,收取3个时间点的细胞总RNA,qRTPCR检测UCA1半衰期。结果:构建的pcDNA6-UCA1过表达质粒转入HEK293T细胞后,qRT-PCR检测UCA1表达水平显著提高;miR-216b能够降低细胞内源或外源过表达的UCA1稳定性,加速UCA1的降解,显著缩短其半衰期;使用miR-216b的抑制剂,UCA1的半衰期延长。结论:miR-216b能够加速lncRNA-UCA1的降解,为研究miRNA与lncRNA的相互作用提供了新的思路。Objective： To explore the effects of miR-216b on lncRNA-UCA1 after miR-216b binding with UCA1.Methods： Genome was extracted from HEK293T cell, and from which UCA1-coding sequence was amplified by PCR with specific primers, then the gene fragment was cloned into pcDNA6.0b vector. Cells were transfected with miR-216b mimic and treated with actinomycin D, then total RNA were extracted at three time points and qRT-PCR was performed to detect the half-life of UCA1. The recombinant pcDNA6-UCA1 was transfected into cells for 24 h, then we transfected miR-216b mimic or miR-216b inhibitor into cells and treated cells with actinomycin D, three time points total cell RNA was harvested, and UCA1 half-life was detected by qRT-PCR.Results： UCA1 RNA level was remarkably increased after transfection of pcDNA6-UCA1. Overexpression of miR-216b significantly reduced the half-life of both endogenous and exogenous UCA1 compared to the control group. miR-216b inhibitor could prolong UCA1 half-life and enhance UCA1 RNA stability.Conclusion： miR-216b could accelerate decay of UCA1 which provided a new thought for interaction between miRNA with lncRNA.

关 键 词：UCA1 长非编码RNA miR-216b 降解 

分 类 号：Q78[生物学—分子生物学]