RNA甲基化修饰在心脏祖细胞来源外泌体治疗心肌梗死中的作用及机制研究

Cardiac progenitor cell-derived exosomes (CPC-Ex) are currently a hot topic in the field of myocardial regeneration after acute myocardial infarction (AMI).In previous studies, we have shown that CPC-Ex can inhibit the apoptosis of cardiomyocytes (CMs). Recently, our preliminary experiments demonstrated that CPC-Ex can cause the upregulation of miR21 and downregulation of RNA methylase METTL3 in hypoxic CMs. However, further study is required to determine the interaction between the two and related anti-apoptotic mechanisms.In this study, we will ① observe the effect of miR21 on CMs in METTL3 expression, methylation level of PTEN mRNA and degree of apoptosis using mimics and inhibitor and determine the interaction between miR21 and METTL3 by dual luciferase reporter gene assay; ② elucidate the role of METTL3-mediated RNA methylation in inhibiting the apoptosis of CMs by miR21 by transgenic and gene knockout techniques; ③ verify that PTEN-PI3K/AKT pathway is involved in the protective effect of miR21 on myocardium through gene silencing and overexpression. We aim to clarify the role and mechanism of RNA methylation in the treatment of AMI by CPC-Ex, providing new therapeutic strategies and targets for AMI treatment.

心脏祖细胞来源外泌体（CPC-Ex）是目前急性心肌梗死（AMI）后心肌再生治疗的研究热点。课题组前期研究发现：CPC-Ex可抑制心肌细胞（CMs）凋亡。近期预实验证实：CPC-Ex可上调缺氧CMs中miR21水平，下调RNA甲基化酶METTL3水平，但两者相互关系及其抗凋亡机制有待进一步研究。本项目拟：①利用mimics和inhibitor观察miR21对CMsMETTL3表达、PTENmRNA甲基化水平及凋亡程度的影响，利用双荧光素酶报告基因明确miR21与METTL3的相互作用；②利用转基因和基因敲除技术阐明METTL3介导的RNA甲基化在miR21抑制心肌细胞凋亡中的作用；③通过基因沉默和过表达方法验证PTEN-PI3K/AKT通路参与了miR21的心肌保护作用。旨在阐明RNA甲基化修饰在CPC-Ex治疗AMI中的作用及机制，为AMI治疗提供新的策略。

N6-甲基腺苷（m6A）是发生于mRNA中腺苷N6位的甲基化修饰，具有多种生理和病理功能。然而，m6A甲基化在缺氧预适应（HPC）中的确切作用仍然未知。在此，我们观察到HPC处理保护H9C2细胞免受H2O2诱导的损伤，上调了总RNA中的m6A水平以及甲基转移酶METTL3、甲基转移酶METTL14和长非编码RNA（lncRNA）H19的表达。METTL3或METTL14的敲除显著逆转HPC诱导的细胞活力、抗凋亡能力和H19表达的增强。甲基化RNA免疫沉淀表明METTL3或METTL14的敲除降低了lncRNA H19中的m6A水平。功能获得实验表明，H19过表达可以部分挽救HPC处理的H9C2细胞中METTL3或METTL14敲低介导的保护降低。RNA结合蛋白免疫沉淀试验表明METTL3和METTL14可以直接与H19结合。我们的研究发现了HPC保护性治疗中转录后调节的新模式。由于METTL3、METTL14和lncRNA H19参与HPC保护，它们可以被视为HPC衍生的心脏康复和治疗方法中潜在生物标志物和治疗靶点。