人NFE2基因上游增强子lncRNA调控NFE2基因转录和K562细胞增殖  

An Enhancer LncRNA Regulates NFE2 Expression and Proliferation in Human Leukemic K562 Cells

作　　者：卢燕飞 曲颂雅 朱晶晶 刘超 王建[1,2] 韩兵社 张俊芳[1,2,3] LU Yan-Fei;QU Song-Ya;ZHU Jing-Jing;LIU Chao;WANG Jian;HAN Bing-She;ZHANG Jun-Fang(Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 201306,China;National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai 201306,China;Marine Biomedical Science and Technology Innovation Platform of Lin-gang Special Area,Shanghai 201306,China)

机构地区：[1]上海海洋大学,水产种质资源发掘与利用教育部重点实验室,上海201306 [2]上海海洋大学,水产科学国家级实验教学示范中心,上海201306 [3]临港新片区海洋生物医药科技创新型平台,上海201306

出　　处：《生物化学与生物物理进展》2024年第1期190-201,共12页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(81770165),临港新片区海洋生物医药科技创新型平台科创基金和上海市教委水产一流学科建设项目资助。

摘　　要：目的转录因子NFE2的异常表达在许多骨髓增殖性肿瘤患者中被观察到,然而造成这种异常的转录调控机制尚不明确,本研究旨在探究参与NFE2转录调控的元件和分子机制。方法首先通过公共数据库中Ch IP-seq数据和ATAC-seq数据预测NFE2基因的潜在增强子元件,并通过双荧光素酶报告实验进行体外验证。随后,通过PRO-seq和GRO-seq数据结合RACE技术克隆这些增强子RNA转录本,经在线编码潜能预测工具分析认为其为lnc RNA,通过RT-q PCR检测该lnc RNA在不同白血病细胞系中和这些细胞诱导分化前后的表达变化及其亚细胞定位。最后,通过慢病毒系统在K562细胞中过表达和敲降该lnc RNA以探究其功能。结果鉴定出调控NFE2转录的3个增强子元件,分别位于NFE2转录起始位点-3.6k,-6.2k和+6.3k区域,这些元件插入NFE2启动子上游均能增强下游萤火虫荧光素酶的表达。克隆出-3.6k增强子负链方向的转录本,将其鉴定为-3.6k-lnc RNA。本研究发现,该lnc RNA在K562、U937和HL-60这3种白血病细胞系中均有一定程度的表达,且均定位于细胞核内。当该lnc RNA在K562细胞中过表达,NFE2水平随之提高,细胞增殖和细胞迁移能力受到抑制;当其被敲降时,NFE2水平相应降低而K562细胞增殖能力随之升高。结论本文鉴定了调控人NFE2基因转录的3个增强子元件和一条增强子lnc RNA转录本,并验证了该lnc RNA对NFE2转录的正调控作用以及对K562细胞增殖能力具有抑制作用。Objective Transcription factor NFE2 was observed abnormal expression in myeloproliferative neoplasm(MPN)patients.However,how NFE2 is transcriptionally regulated remains ambiguous.This study aims to explore the elements and molecular mechanisms involved in the transcriptional regulation of NFE2.Methods Active enhancers were predicted by public NGS data and conformed experimentally via dual luciferase reporter assay.After that,PRO-seq and GRO-seq data was used to detect enhancer RNAs transcribed from these enhancers.RACE was utilized to clone the full length enhancer RNA(eRNA)transcripts,and RT-qPCR was used to measure their expression in different leukemia cell lines as well as the transcript levels during induced differentiation.Finally,to investigate the molecular function of the eRNA,overexpression and knockdown of the eRNA via lentivirus system was performed in K562 cells.Results We identified three enhancers regulating NFE2 transcription,which located at-3.6k,-6.2k and+6.3k from NFE2 transcription start site(TSS)respectively.At the-3.6k enhancer,we cloned an eRNA transcript and characterized that as a lncRNA which was expressed and located in the nucleus in three types of leukemia cell lines.When this lncRNA was overexpressed,expression of NFE2 was upregulated and decreases of K562 cell proliferation and migration ability were observed.While knocking down of this lncRNA,the level of NFE2 decreases correspondingly and the proliferation ability of K562 cells increases accordingly.Conclusion We identified an enhancer lncRNA that regulates NFE2 transcription positively and suppresses K562 cell proliferation.

关 键 词：NFE2基因 增强子 长链非编码RNA 细胞增殖 

分 类 号：Q291[生物学—细胞生物学]