长链非编码RNA-Zfas1在心肌梗死中的调控机制研究

Myocardial infarction is a major cause of cardiac sudden death worldwide. The motality of it is still high due to no sensitive biomarker and effective treatment. So it need to explore the mechanism of myocardial infarction further and find new traget molecular. Recently, a new class of non-coding RNAs, long non-coding RNAs (lncRNAs) has been identified in a variety of eukaryotes. Although most remain functionally uncharacterized biological "dark matter," lncRNAs have garnered considerable attention for their diverse roles in human biology. Now more researches have revealed that long non-coding RNA (lncRNA) play an important role in cardiovascular disease. Our previou study has shown that the expression of a lncRNA-Zfas1 is incresed significantly in myocardial tissue of myocardial infarction. Also transduction of cardiomyocytes by reconbination the lentiviral vector in co-expressing Zfas1 and green fluorescent protein (GFP) could impair the cardiac function and cause calcium overload. Further forecast by network has shown that 6 transcription factors and 7 microRNAs have potential to regulate the transcription of Zfas1 gene. Moreover, Zfas1 has a potential to regulate the expression of 5 target gene ranging from ion channel to apoptoisis. Therefore, over-expression of Zfas1 is an important reason for electrophysiological imbalance and cardiomyocyte damage. Based on these results, we will use a serious of molecular biological and electrophysiological techniques to carry out the following research: (1) the regulatory network of miRNA/transcription factor-Zfas1-target gene in myocardial infarction. (2) application of gene silencing technology based on Zfas1 to prevent and cure myocardial infarction. (3) to determine the expression charactor of Zfas1 and ZF-miniRNA in different components of peripheral blood, and to establish an early warning system of myocardial infarction.

心肌梗死是心源性猝死的主要原因，并且长链非编码RNA（lncRNA）在心血管疾病中发挥重要作用。本项目初步研究结果显示心肌梗死时心肌组织中一种lncRNA-Zfas1的表达水平显著增加，同时于心肌在体导入Zfas1重组慢病毒，小鼠心功能显著降低、心肌细胞出现钙超载。通过网络预测有6个转录因子和7个miRNA具有调控Zfas1基因的转录潜力，同时Zfas1具有调控涉及到离子通道、细胞凋亡等5个靶基因表达的潜力，因此Zfas1过表达是心肌梗死时电生理失衡和细胞损伤的重要原因。本项目将应用电生理学、分子生物学等手段阐明（1）心肌梗死时miRNA/转录因子-Zfas1-靶基因的调控网络；（2）首次以Zfas1为靶点应用基因沉默技术对心肌梗死进行预防和治疗，为心肌梗死的药物及基因治疗提供新思路和新措施；（3）确定外周血不同成分中Zfas1及ZF-miniRNA的表达特征，建立心肌梗死的早期预警体系。

心肌梗死是心源性猝死的主要原因，给社会和家庭造成了巨大的打击和沉重的负担。因此，发现新的心肌梗死预警因子，揭示心肌梗死的病理生理学新机制，具有重要的社会意义。在本项目的资助下，我们研究发现lncRNA-ZFAS1是心肌梗死发生和发展的重要调控因子。首先我们发现lncRNA-ZFAS1在心肌梗死心脏组织中和缺氧处理的心肌细胞中其表达水平显著升高，并且原位杂交实验结果显示，lncRNA-ZFAS1在细胞核和细胞质中均有分布。在细胞水平上，lncRNA-ZFAS1过表达可以减弱心肌的收缩功能，敲减lncRNA-ZFAS1可以缓解心肌梗死模型小鼠心脏收缩舒张功能的损伤。在亚细胞层面，lncRNA-ZFAS1能够负性调控心肌细胞以及心脏整体内钙瞬变，引起心肌细胞内钙超载，干扰心肌细胞内钙稳态。在分子层面，lncRNA-ZFAS1可以特异性地与SERCA2a蛋白直接结合，并抑制该蛋白的表达和功能。进一步研究发现，lncRNA-ZFAS1序列中存在一段功能序列（ZFAS1-FD），该功能序列在人和小鼠之间高度保守，并且具有与lncRNA-ZFAS1全长序列相似的功能：与SERCA蛋白结合，抑制SERCA2a蛋白和mRNA的表达，影响心肌细胞内钙瞬变和静息钙，调控钙稳态。当突变该功能序列中与SERCA蛋白结合的位点后，得到mut-ZFAS1-FD，该突变序列对心肌细胞中SERCA2a蛋白的功能和表达均无显著影响。而lncRNA-ZFAS1功能序列的反义链可以抑制lncRNA-ZFAS1全长对心肌细胞的损伤性作用。同时研究发现，NFATC2是lncRNA-ZFAS1上游的调控因子，是引起心肌梗死时lncRNA-ZFAS1表达上调的直接原因。结论及科学意义：本研究首次发现lncRNA-ZFAS1具有成为急性心肌梗死外周血生物诊断标志物的潜力，为心肌梗死早期预警体系的建立提供了新的标志物。同时，lncRNA-ZFAS1在心肌梗死心脏组织中的高表达是引发心肌梗死后心脏功能障碍的重要原因，因此，本研究所建立的NFATC2- lncRNA-ZFAS1-SERCA2a钙稳态调控网络为心肌梗死的药物及基因治疗提供新思路和新措施。