微小RNA-409-3p在血浆外泌体抑制心肌纤维化中的作用及机制研究

Myocardial fibrosis is a common pathological change in many cardiac diseases. Exosomes can participate in the pathophysiological process of the heart by carrying nucleic acids, proteins and other bioactive substances. MicroRNA is a single-stranded non-coding RNA composed of 20-23 nucleotides, which plays an important role in the development of cardiac diseases. MicroRNA-409-3p, a member of the mature microRNA-409 family, is considered as a tumor suppressor. Some studies suggest that it can be used as a diagnostic marker of atrial fibrillation and heart failure. No studies have shown that it is related to myocardial fibrosis. LRP6 is a common receptor of Wnt/β-catenin signaling pathway, it can be recruited to promote activation of cell signal. Preliminary experiments showed that plasma exosomes of healthy people could inhibit fibroblast fibrosis, and microRNA-409-3p in plasma exosomes of patients with myocardial fibrosis was significantly reduced. Bioinformatics predicted that LRP6 was the target gene of microRNA-409-3p which could inhibit myocardial fibrosis and inhibit the expression of LRP6 protein in our preliminary experiment results. Therefore, we propose a scientific hypothesis on the basis of preliminary experiments: plasma exosomes inhibit LRP6 through microRNA-409-3p, and affect Wnt/β-catenin pathway to play an anti-myocardial fibrosis role. The aim of this project is to explore the role and molecular mechanism of microRNA-409-3p in the anti-myocardial fibrosis effect of plasma exosomes, composing of functional deletion and acquired experiments in vitro and in vivo, so as to provide a possible new target for clinical prevention and treatment of myocardial fibrosis.

心肌纤维化是多种心脏疾病共同病理改变。外泌体携带核酸、蛋白等参与心脏病理过程。微小RNA是由20-23个核苷酸组成的单链非编码RNA，在心脏疾病有重要作用。微小RNA-409-3p是成熟微小RNA-409家族成员，被认为是肿瘤抑制因子，有研究提示其可作为房颤和心衰的诊断标志物，尚无研究表明其与心肌纤维化有关。LRP6是Wnt/β-catenin信号通路共同受体，被招募到Wnt受体促进信号激活。预实验发现健康人血浆外泌体可抑制成纤维细胞纤维化，心肌纤维化患者血浆外泌体中微小RNA-409-3p明显降低，生物信息学预测LRP6为其靶基因，并可抑制成纤维细胞纤维化，且下调LRP6蛋白表达。本项目拟在细胞和动物两个层面，结合功能缺失性和获得性实验来验证科学假说：血浆外泌体通过微小RNA-409-3p抑制LRP6，减弱Wnt/β-catenin通路发挥抗心肌纤维化作用，为防治心肌纤维化提供新靶点。

心肌纤维化是众多导致心力衰竭的慢性心血管疾病进展到终末阶段的标志，然而目前对其尚无有效的治疗方法，且发生机制还不明确。微小RNA失调已被证实参与心肌纤维化的发生发展，本研究是首次探索微小RNA-409-3p在心肌纤维化中的作用及分子机制。我们发现微小RNA-409-3p在三种纤维化模型中均增加：包括慢性心肌梗死小鼠的心脏组织、用血管紧张素II或转化生长因子-β处理的新生大鼠心脏成纤维细胞。体内实验结果表明慢性心肌梗死手术诱导的心肌纤维化和心功能障碍可因系统性敲低微小RNA-409-3p而减轻。体外实验结果表明转染微小RNA-409-3p模拟物可促进心脏成纤维细胞增殖并向肌成纤维细胞分化，同时伴随Col1a1、Col3a1和α-SMA的蛋白表达量上调；而微小RNA-409-3p抑制剂则表现出相反的效果。本研究还进一步验证了Gpd1是微小RNA-409-3p的直接作用靶点，功能挽救实验结果显示Gpd1 siRNA消除了微小RNA-409-3p抑制剂在新生大鼠心脏成纤维细胞中的抗纤维化作用，说明微小RNA-409-3p通过Gpd1发挥促进心肌纤维化的作用。此外，我们还鉴定出GATA2是微小RNA-409-3p在心肌纤维化相关通路中的上游正转录因子。本研究表明调节微小RNA-409-3p或许可成为治疗心肌纤维化的潜在新方法。