miR-144/451的体外红系发生示踪揭示LINC01569的潜在红系发生调控影响  

The in vitro tracing of miR-144/451 reveals the potential regulatory function of LINC01569 in erythropoiesis

作　　者：廖炳懿 孙文翠[1] 唐诗丽 黄恩霞 刘晴蓉 薛原 张勇刚 LIAO Bingyi;SUN Wencui;TANG Shili;HUANG Enxia;LIU Qingrong;XUE Yuan;ZHANG Yonggang(Institute of Blood Transfusion,Chinese Academy of Medical Sciences&Peking Union Medical College,Chengdu 610052,China)

机构地区：[1]中国医学科学院北京协和医学院输血研究所,四川成都610052

出　　处：《中国输血杂志》2024年第5期516-523,共8页Chinese Journal of Blood Transfusion

基　　金：成都市科技局重点研发支撑计划技术创新研发项目(2022-YF05-02069-SN)。

摘　　要：目的用已构建的miR-144-GFP-H1人胚胎干细胞(human embryonic stem cell,hESC)示踪细胞株,以GFP荧光信号报告miR-144表达描述红系发生的进展程度,探究在红系发生过程中lncRNA的潜在功能并初步验证其功能影响。方法利用已构建的miR-144/451-GFP-H1细胞株(下文简称144-H1)进行体外红细胞诱导培养。以CD71,GPA表面分子描述进入红系发生阶段的细胞亚群。以miR-144的GFP报告基因为关键区分,对GFP阳性(高红系发生倾向)和阴性细胞群(低红系发生倾向)分别进行转录组测序,获取差异性表达的lncRNA。选取具备验证价值的lncRNA条目进行初步功能验证。利用CRISPR/Cas9基因编辑技术设计对应lncRNA的功能干扰方案并获取对应lncRNA功能敲除的144-H1细胞株。使用该敲除细胞株与非敲除的144-H1细胞株进行红细胞的平行诱导培养,寻找差异节点,初步验证其对在体外发育模型下对红系发生的影响效应。结果1)构建的144-H1细胞株能够在进入体外红细胞诱导阶段后表达GFP荧光蛋白,提示miR-144的启动。2)在CD71,GPA双阳群中,GFP阳性亚群和GFP阴性亚群存在显著的lncRNA表达差异。3)对多个lncRNA条目设计了能够有效干扰其功能的序列区段删除基因编辑方案。验证编辑成功。在敲除细胞株中,lncRNA的一部分序列被直接删除。4)在红细胞诱导培养的平行验证试验中,LINC01569的功能敲除株对比非敲除株表现出明显的流式亚群差异和细胞增殖能力差异:①敲除株的GFP荧光持续高表达;②敲除株CD71-GPA双阳群在红细胞成熟过中比例持续降低;③敲除株未观察到显著的血红蛋白表达,无明显红色;④敲除株细胞增殖能力远低于非敲除株(P<0.05)。结论成功利用144-H1细胞株对lncRNA在红系发育中的潜在功能进行了探究,可设计更精密的体外发育实验来提高lncRNA功能的抓取精度。在差异性表达的lncRNA条目中,LINC01569经初步验证,对红系发生�Objective Utilizing a specially engineered miR-144-GFP-H1 human embryonic stem cell(hESC)reporter line,this study leverages GFP fluorescence as an indicator of miR-144 expression to gauge the progression of erythropoiesis.The investigation is aimed at elucidating the potential roles of lncRNAs within the erythropoietic framework and conducting an initial assessment of their functional impact.Methods The miR-144/451-GFP-H1 cell line(hereafter referred to as 144-H1)was utilized for in vitro erythrocyte induction culture.The subpopulations of cells entering the erythropoiesis stage were characterized by the surface molecules CD71 and GPA.The GFP reporter gene of miR-144 served as a critical determinant to distinguish between GFP-positive cells(with a high propensity for erythropoiesis)and GFP-negative cells(with a low propensity for erythropoiesis).Transcriptome sequencing was performed on both groups to identify differentially expressed long non-coding RNAs(lncRNAs).LncRNA entries with potential for validation were selected for preliminary functional verification.The CRISPR/Cas9 gene editing technique was employed to design functional interference strategies for the targeted lncRNAs,obtaining 144-H1 cell lines with knocked-out function of the specific lncRNAs.These knockout cell lines,along with non-knockout 144-H1 cell lines,were used for parallel erythrocyte induction culture to identify differential nodes.This approach preliminarily verified their impact on erythropoiesis in an in vitro development model.Results 1)The constructed 144-H1 cell line was capable of expressing GFP fluorescence upon entering the stage of in vitro erythrocyte induction,indicating the activation of miR-144/451.2)Within the CD71,GPA double-positive group,significant differences in lncRNA expression were observed between the GFP-positive and GFP-negative subpopulations.3)Gene editing strategies involving the deletion of sequence segments capable of effectively interfering with the function of multiple lncRNA entries were designed and veri

关 键 词：miR144/451 人胚胎干细胞 长链非编码RNA 红系发生 

分 类 号：R457.1[医药卫生—治疗学]