miR-146b-5p参与调控TIMP-4/MMP-9在心房颤动心房纤维化机制中的研究

Atrial fibrillation (AF) is the most common sustained cardiac rhythm disorder, and is associated with pronounced morbidity, mortality, and socio-economic burden. An important acknowledgement is that AF atrial structure remodeling in a way that promotes its occurrence and maintenance. Matrix synthesis and degradation is mainly regulated by the balance between matrix MMPs and tissue inhibitors of the TIMP axis. In the heart, MMP-9 and TIMP-4 are predominantly involved in cardiac remodeling. Evidence has proved MMP-9 remains latent but is activated in certain pathological conditions and degrades the matrix, resulting in the deposition of oxidized collagen and leading to fibrosis; TIMP-4 mitigates the effect of MMP-9. MicroRNAs (miRNAs) are ~22-nucleotide single-stranded RNAs that inhibit the expression of specific mRNA targets through Watson–Crick base pairing between the miRNA ‘seed region’ and sequences commonly located in the 3'-untranslated regions. Although the cellular mechanisms and gene mutations responsible for numerous cardiovascular disorders have been extensively studied, it has become apparent only recently that miRNAs have key roles in cardiovascular development and disease. In our previous study, we identified 10 differentially expressed miRNAs and 624 differentially expressed mRNAs, among which only one miRNA-target gene pair miR-146b-5p and TIMP-4 was constructed. The validated results revealed that miR-146b-5p, MMP-9 and collagen content were upregulated, whereas TIMP-4 was downregulated in the atrial fibrillation patients. After transfection of miR-146b-5p into the cardiac fibroblasts, TIMP-4 expression was markedly reduced and collagen content was increased. Moreover, the luciferase results confirmed that TIMP-4 was a target of miR-146b-5p. This work reveals miR-146b-5p probably acts as an intracellular mediator via TIMP-4/MMP-9 in the maladaptive remodeling of atrial fibrosis in atrial fibrillation. In this application, we are going to test this hypothesis. We plan to combine in vitro AF cellular model of human atrial myocytes differentiated from human embryonic stem cells and animal model to assess (1) whether miR-146b-5p contributes balance between TIMP-4 and MMP-9 and collagen metabolism in vitro AF cellular model; and (2) whether downregulation of endogenous miR-146b-5p could reduces atrial fibrosis and prevent structure remodeling with a atrial fibrosis mouse model. .The accomplishment of this application will provide a new understanding of underlying mechanisms of atrial structure remodeling to allow for the definition of novel molecular targets in AF. And it is tempting to pave the avenue for more specific novel drug targets and potential for developing that missing pharmacopeia for AF.

心房纤维化在心房颤动（房颤）的发生和发展过程中占有重要地位，TIMPs/MMPs失调是心房纤维化最主要的分子基础。miRNA作为科学界研究的热点，在心血管系统中的关键调控作用不断得以阐明。本项目组前期发现房颤患者左心耳组织miR-146b-5p的表达上调和预测的靶基因TIMP-4表达下调；体外构建报告载体等实验证实miR-146b-5p可以结合到TIMP-4的3'-UTR区，而且过表达miR-146b-5p使TIMP-4蛋白表达降低，MMP-9和胶原蛋白表达增高，提示miR-146b-5p有可能通过调控TIMP-4/MMP-9参与了组织重构。本研究拟利用人胚胎干细胞分化的心房肌细胞建立房颤细胞模型，结合动物模型，深入探讨miRNA调控心房纤维化的机制。本研究不仅对阐明miRNA调控心房纤维化的机制有重要意义，而且为寻找可逆转心房纤维化的新靶点奠定了理论基础，为房颤的上游治疗提供了新的思路。

心房颤动(房颤) 是临床上最常见的心律失常， 并且有很高的致残率和致死率，严重威胁人类的生命和健康。心房纤维化重构促使房颤发生和延续， 改善心房纤维化可以有效预防房颤。微RNA (miRNA)是新发现的基因表达调控因子，研究已表明其通过参与调控纤维化相关蛋白的表达，参与瓣膜性房颤的发生和发展，但是作为绝大多数的非瓣膜性房颤，其机制和miRNA的关系的研究却未见报道。本研究目的是基于前期非瓣膜房颤心房组织芯片研究的重要发现—miR-146-5p，阐释其通过调节靶基因TIMP-4，进一步导致MMP-9和胶原蛋白表达异常，参与房颤心房纤维化的调控机制。研究通过人胚胎干细胞衍生的心房肌细胞起搏膜型，犬房颤模型和小鼠的心肌纤维化模型，从细胞分子水平和动物实验探讨miR-146-5p通过调控TIMP-4，产生心房纤维化的机制。.首先利用视黄酸信号调控人胚胎干细胞分化为经验证的人源性的心房肌细胞，转染过表达miR-146后，建立快速细胞起搏模型，PCR和WB检测显示TIMP-4 mRNA和蛋白表达水平显著下调，而MMP-9和胶原蛋白 mRNA和蛋白表达水平显著上调。其次利用比格犬建立房颤模型，结果显示房颤组TIMP-4 mRNA和蛋白表达水平显著抑制下调，伴随MMP-9和胶原蛋白 mRNA和蛋白表达水平显著上调，使得心房纤维化。而在小鼠心肌纤维化模型中，与假手术组相比较，PCR和WB检测显示TIMP-4 mRNA和蛋白表达水平显著下调，而MMP-9和胶原蛋白 mRNA和蛋白表达水平显著上调。.由此可见miR-146-5p通过在转录后水平调控TIMP-4蛋白表达，进而破坏TIMPs/MMPs平衡，使得MMP-9和胶原蛋白表达异常，影响了心房的组织重构，产生纤维化，最终导致了房颤的发生和发展。本研究不仅对阐明miR-146-5p调控心肌细胞纤维化的机制有重要意义，而且为寻找可逆转纤维化的新靶点奠定了理论基础，为房颤的上游治疗提供了新的思路。