MicroRNA调控斑马鱼心脏损伤后再生的作用和机制

Because the mature cardiomyocytes hardly exist differentiation and proliferation, the heart can not repair itself by regeneration in the face of myocardial injury, such as myocardial infarction, resulting in the loss of part of the cardiac function and even heart failure. The discovery of zebrafish heart regeneration provides a research platform for further exploring the myocardial cells differentiation and proliferation, but the underlying mechanism of zebrafish cardiac regeneration is still unclear. Our preliminary experiments showed that the expression of many kinds of miRNAs had evident change during the regeneration process after the damage of adult zebrafish heart. Intervention with specific miRNA could influence the proliferation of cultured zebrafish cardiomyocytes. Therefore, the aim of this study is to: 1) make it clear that which miRNAs' expression will change significantly in zebrafish heart regeneration process by chip analysis and quantitative PCR method; 2) observe the effect of specific miRNA on cardiomyocyte proliferation and cardiac regeneration in vitro and in vivo by knock-down and over-expression of the level of miRNAs; 3) identify the functional target gene of specific miRNA, which may play a key role in the zebrafish heart regeneration process. In-depth study of the role and mechanism of miRNAs in cardiac repair and regeneration after injury using zebrafish heart regeneration model will provide specific miRNA as an intervention target to treat heart injury for cardiac regeneration repair.

成熟的心肌细胞几乎不存在分化、增殖的能力。因此，心脏在损伤情况下（如心肌梗死）不能进行自身再生修复，从而导致心脏功能的部分丢失甚至衰竭。斑马鱼心脏再生能力的发现，为进一步探究心肌细胞分化、增殖提供了研究平台，但其再生机制仍不清楚。课题组前期实验显示，成年斑马鱼心脏损伤后再生过程中多个microRNA(miRNA)的表达变化显著，干预特定miRNA对培养的斑马鱼心肌细胞增殖能力有明显影响。本研究拟：1) 通过miRNA芯片、定量PCR等方法明确斑马鱼心脏再生过程中表达明显变化的miRNA；2) 在离体和整体水平，通过基因干预的方法确定特定miRNA对心肌细胞增殖和心脏再生能力的影响；3）阐明特定miRNA在斑马鱼心脏再生过程中起关键作用的分子机制。本项目利用斑马鱼心脏再生模型深入研究miRNA在心脏损伤后修复中的作用和机制，为以特定miRNA作为干预靶点对心脏的再生修复治疗开辟一条重要的途径

本项目构建了斑马鱼心脏再生模型，并对切除面积，斑马鱼鱼龄等参数进行了优化，当MS-222浓度为40mg/L，心室切除面积为20%时，斑马鱼入麻及苏醒较为平稳，术后斑马鱼复苏时间较短，且保持了较高的术后生存率。完成了斑马鱼心脏再生模型中microRNA表达谱的高通量测序及PCR验证，提示microRNA-301c-5p下调最为明显；microRNA-21-5p上调最为明显。建立乳鼠心肌细胞的原代培养方法，通过低氧诱导心肌细胞增殖，重新验证高通量测序结果及靶基因预测结果，发现MicroRNA-21-5p 水平的上调与Alx1和Pja2的下调具有时间相关性。通过双荧光素酶报告基因实验证实microRNA-21-5p在离体体系中能够调控Alx1和Pja2的表达。设计并合成microRNA-21-5p的mimic和inhibitor、 Pja2和Alx1的siRNA，转染乳鼠原代心肌细胞并验证其过表达和沉默效率，以EDU作为心肌细胞增殖指标，通过免疫荧光实验检测乳鼠心肌细胞增殖水平的变化。在乳鼠心肌细胞增殖过程中，microRNA21-5p可调控Pja2的表达，并促进心肌细胞增殖水平；而直接敲低Pja2对心肌细胞再生也有明显促进作用，但对microRNA21-5p的表达没有明显影响。MicroRNA-21-5p对心肌再生的促进作用可能是通过抑制Pja2表达实现的，而Alx1虽受microRNA-21-5p的调控，但并不参与心脏再生过程。项目培养博士1名，硕士1名，发表SCI论文1篇，影响因子6.086，中文期刊4篇，其中核心期刊论文1篇。