miR-26负向调控AngII/KLF4/TGF-β通路对心房纤维化和房颤易感性的影响及其机制研究

Atrial fibrosis serves as an important substrate for the initiation and maintenance of atrial fibrillation (AF). Recent findings suggest that multiple microRNAs（miRs）are involved in the process of atrial fibrosis via targeting the transforming growth factor β (TGF-β) signaling pathway. Our preliminary findings indicated that miR-26a/b, which is abundantly expressed in heart tissue, suppressed atrial fibrosis and AF initiation, as well as down-regulated the expressions of TGFβ1 and TGFβRI. However, the precise regulatory mechanisms remain unknown. Therefore, in the present study we sought to examine the regulatory effects of miR-26a on atrial fibrosis and AF susceptibility, and to elucidate the underlying mechanisms. We aim to: 1) reveal the potential correlations between atrial fibrosis and the expressions of miR-26a/b, Kruppel-like factor 4 (KLF4) and key components of Ang II/TGF-β pathway in patients with AF; 2) explore the effects of miR-26a on AF susceptibility, atrial fibrosis, KLF4 expression and Ang II/TGF-β signaling, through specific up- or down-regulation of miR-26a [using miR-26a-expressing adenovirus (Adv-miR-26a) or locked nucleic acid anti-miR-26 (LNA-anti-miR-26a)] in a murine AF model [induced by continuous Ang II infusion]; 3) determine the effects of miR-26a on fibroblast pathophysiology, including proliferation, differentiation and collegen synthesis, as well as to elucidate the molecular mechanisms through which miR-26a suppresses atrial fibrosis by targeting Ang II/KLF4/TGF-β pathway at multiple levels. Collectively, our study would uncover new insights into the pathogenesis of AF, and provide novel evidence for potential therapeutic approaches targeting atrial fibrosis.

心房纤维化是房颤发生与维持的重要基质。多种miRs能够通过调控TGF-β通路参与心房纤维化过程。我们的前期研究显示，miR-26a抑制小鼠房颤与心房纤维化的同时，亦下调TGFβ1与TGFβRI的表达，但其具体机制不明。本课题拟①观察房颤患者心房组织纤维化改变与miR-26、KLF4和Ang II/TGF-β通路相关分子表达的关联；②构建AngII诱导的小鼠心房纤维化-房颤模型，在体观察上调或阻断miR-26a后房颤诱发和持续、心房纤维化指标、KLF4和Ang II/TGF-β通路关键分子的表达情况，明确miR-26a在房颤易感性和心房纤维化中的作用；③离体观察miR-26a对心脏成纤维细胞功能的影响，阐明miR-26a通过多靶点调控Ang II/KLF4/TGF-β通路抑制心房纤维化的分子机制。本课题可望阐明miR-26在心房纤维化中的作用与机制，为房颤靶向治疗提供新的理论基础和实验依据。

心房颤动（房颤、AF）是临床工作中最常见的心律失常，其导致心房收缩功能丧失、心功能受损，同时易化心房血栓形成、机体重要器官栓塞，显著增加了心血管疾病的致死率和致残率。其中，心房纤维化是房颤心房结构重构的主要表现形式，也是众多致房颤病因的共同病理基础，深入研究心房纤维化的病理机制，积极寻找有效的干预靶点是目前房颤研究领域的焦点。既往研究显示，miR-26a具有抗房颤电重构效应；然而，其在心房纤维化中的作用及机制仍待阐明。.本项目通过临床/动物/细胞实验、干预研究等旨在从临床/基础、体内/体外，临床/动物/分子生物学等层面详细阐明miR-26a抗心房纤维化效应及其作用机制。.通过取材/分析房颤患者心房组织：房颤患者心房纤维化的程度较窦性心律者显著增高，miR-26a/b表达下调，而Ang II/TGF-β信号通路关键分子、KLF4以及纤维化相关指标表达上调，miR-26a/b表达水平与心房纤维化、Ang II/TGF-β信号通路关键分子以及纤维化相关指标成负相关。通构建血管紧张素II-房颤（Ang-II AF）小鼠模型，以及分离培养小鼠/人体心脏成纤维细胞（CF）并进行miR-26干预： Ang II上调能够显著增加房颤易感性，同时诱导心房组织、CF miR-26a/b表达下调，Ang II/KLF 4/TGF-β通路激活、通路关键分子表达上调，CF分化、增殖、迁移活性增强以及胶原纤维蛋白分泌增加；而转染miR-26a拟似物(miR-26a mimic)显著抑制了CF Ang II/KLF4/TGF-β通路激活、通路关键分子的表达，以及细胞分化、增殖、迁移活性增强以及胶原蛋白的表达。通过生物信息学以及分子生物学分析结果显示，miR-26通过靶向负调控KLF4，间接抑制CF TGF-β信号通路、进而发挥抗房颤纤维化效应.miR-26抗房颤电重构作用的同时，具备抗心房结构重构效应：其通过靶向负调控KLF4基因表达，间接抑制心脏成纤维细胞TGFβ1与TGFβRI的表达；miR-26可通过直接或间接作用，同时调控TGF-β信号通路中包括TGFβ1/TGFβRI/Smad4/CTGF和CoL I在内的多个靶点，阻断心脏成纤维细胞TGF-β通路激活，进而抑制心脏成纤维细胞分化、迁移、增殖活性以及胶原蛋白分泌，从而于多个层面发挥抗房颤、抗心房纤维化效应，发挥负性调控心房纤维化效应。