核仁素通过调控lncRNA-FENDRR保护心肌缺血-再灌注损伤的机制研究

Nucleolin is a multifunctional RNA-binding protein that exhibits an unusual range of activities. However, whether nucleolin plays roles in regulation of lncRNAs expression or not is still unclear. Our previous studies showed that nucleolin protects myocardium against ischemia-reperfusion injury. At present,we have screened the nucleolin target lncRNAs by lncRNA microarray analysis in nucleolin overexpression cardiomyocytes after hydrogen peroxide exposure, and 276 different expression of lncRNAs were found, and eight lncRNAs including lncRNA-FENDRR were found can interact with nucleolin.Furthermore,lncRNA-FENDRR was found contain 3 nucleolin recognition element (NRE)in its sequence.So we hypothesized that nucleolin might regulate the expression of lncRNA-FENDRR through binding to its NRE and regulating its stability, then plays cardiomyocyte protective role. In this project, we will study the expression and role of lncRNA-FENDRR in cardiomyocyte injury mediated by hydrogen peroxide by using full-length FENDRR plasmid and siRNA against FENDRR, and the effect of nucleolin on expression of lncRNA-FENDRR. we will also approach the molecular mechanism by which nucleolin regulates lncRNA-FENDRR by using Bioinformatics, RNA Transcription, RNA pull-down assay, coimmunoprecipitation of protein and RNA, and luciferase reporter assay. Carrying out this project can help us to explain the effect of nucleolin on myocardial protective role and its new molecular mechanism and to provide new ideas and experimental clues for prevention and cure of related deseases.

核仁素是具有多种生物学功能的RNA结合蛋白，但核仁素是否调控lncRNA表达，是一全新研究领域。申请者采用核仁素过表达心肌细胞，通过lncRNA芯片发现267个差异表达lncRNAs，进一步发现其中lncRNA-FENDRR等8个lncRNA可与核仁素相结合，而且lncRNA-FENDRR序列中含多个完整的核仁素结合元件，推测核仁素可能通过与lncRNA-FENDRR结合，调控其表达，进而起心肌保护作用。本项目拟在此基础上，采用心肌缺血-再灌注损伤动物模型及氧化应激细胞模型探讨lncRNA-FENDRR的生物学意义；采用转基因小鼠及RNA干扰探讨核仁素对lncRNA-FENDRR表达的影响；采用RNA pull-down、蛋白质-RNA免疫共沉淀及稳定性分析等探讨核仁素调控lncRNA-FENDRR的机制。开展本项目有助于阐释核仁素抗心肌损伤的新机制，为心肌保护提供新思路和实验线索。

核仁素是一种核仁蛋白质，具有多种生物学功能的RNA结合蛋白。长链非编码 RNA（lncRNA）是一类长度大于200个核苷酸，缺少开放阅读框且无蛋白编码能力的内源性RNA分子。申请者采用核仁素过表达心肌细胞，通过lncRNA芯片发现267个差异表达lncRNAs，进一步发现其中lncRNA-FENDRR等8个lncRNA可与核仁素相结合，而且lncRNA-FENDRR等多个lncRNA序列中含多个完整的核仁素结合元件，推测核仁素可能通过与lncRNA-FENDRR结合，调控其表达，进而起心肌保护作用。在此基础上本项目采用心肌缺血-再灌注损伤动物模型及氧化应激细胞模型探讨lncRNA-FENDRR的生物学意义；采用转基因小鼠及RNA干扰探讨核仁素对lncRNA-FENDRR表达的影响；采用RNA pull-down、蛋白质-RNA免疫共沉淀及稳定性分析等探讨核仁素调控lncRNA-FENDRR的机制。结果显示：1、心肌细胞中lncRNA FENDRR等多个lncRNAs的表达可受核仁素调控；2、FENDRR在心肌缺血-再灌注损伤及阿霉素所致心肌损伤中表达下调，发挥心肌保护作用；3、核仁素可通过结合FENDRR，调控FENDRR表达，在氧化应激损伤中发挥心肌细胞保护作用；4、lncRNA H19在心肌缺血预适应体内及体外实验中表达上调；5、lncRNA H19可与核仁素相互作用，增加核仁素蛋白的稳定性，上调核仁素表达，在心肌缺血预适应发挥保护作用；6、lncRNA CARMN在心肌梗死后心脏组织中表达上调；7、核仁素可与lncRNA CARMN的结合，下调CARMN的表达，在心肌梗死后发挥促血管新生作用。本研究发现核仁素可通过调控lncRNA表达在心肌损伤发挥保护作用，为心肌保护研究开辟了新的领域，也为心肌损伤防治提供线索和新思路。