miRNA-215、miRNA-199在心房颤动心房Cx40重构中的作用研究

Some evidences have indicated that upregulation of miRNA-215 and miRNA-199 contributes to atrial fibrillation (AF). However, the mechanism of miRNA-215 and miRNA-199 involving in the pathogenesis of AF is unknown. Our previous studies show that Cx40 remolding plays an important role in AF. Nkx2.5 encoded by NKX2-5, a transcription factor, activates the transcription of GJA5 by binding its promoter. Interestingly, the NKX-2-5 and GJA5 encoding Cx40 are potential target genes for miRNA-215 and miRNA-199, respectively. We propose the hypothesis that miRNA-215 and miRNA-199 may take part in the pathogenesis for AF by regulating Cx40 in transcriptional and posttranscriptional level, respectively. To testify this hypothesis, The expression of miRNA-215，miRNA-199, Nkx2.5 and Cx40 will be measured in the patients with AF and the rapid atrial pacing canine models by RT-qPCR, immunofluorescence and Western-blot. The human atrial myocyte and mouse models of miRNA-215/miRNA-199 overexpression or underexpression will be made by miRNA mimic/inhibitor transfection and lentiviral vector transfection, respectively. The effect of miRNA-215/miRNA-199 overexpression or underexpression on the Nkx 2.5, Cx40 and AF susceptibility will be observed. Therefore, the effect of miRNA-215 and miRNA-199 on atrial Cx40 remolding in atrial fibrillation will be investigated to reveal the pathogenesis of AF and provide new targets for the intervention of AF.

研究表明miRNA-215和miRNA-199可能参与心房颤动（房颤）的发生，但机制尚不明确。我们前期的工作提示心房Cx40重构与房颤发生密切相关。Nkx2.5(Cx40转录因子)和Cx40基因分别是miRNA-215、miRNA-199潜在靶基因。据此，我们假说，各种病因导致的miRNA-215、miRNA-199上调，在转录及转录后水平下调Cx40，参与房颤的发生。我们将利用PCR、免疫荧光、Western-blot、双荧光素酶报告基因和基因干预等方法，检测房颤患者和房颤犬心房miRNA-215、miRNA-199、Nkx2.5及Cx40表达变化；构建miRNA-215、miRNA-199过/低表达心房肌细胞和小鼠模型，观察对Nkx2.5、Cx40表达和房颤易感性的影响，探讨miRNA-215、miRNA-199对心房Cx40重构的调控机制及其在房颤发生中的作用，为房颤干预提供新靶点。

本项目首次探索了miR-199及miR-215在房颤心房Cx40/Nkx2.5重构作用以及miR-223-3p靶向调控NLRP3介导Ang II/ROS/NLPR3炎症小体信号通路参与房颤机制。我们发现，房颤患者右心房Cx40蛋白表达水平明显下降，房颤组miRNA-199、miRNA-215表达呈上升趋势，而Nkx2.5呈下降趋势，这与课题提出的科学假说相符，提示miRNA-199、miRNA-215可能分别通过调控Cx40和Nkx2.5参与房颤的发生。然后，我们成功利用氯化钙联合乙酰胆碱诱导SD大鼠产生房颤，并以此发现房颤组大鼠miR-199表达升高，合并大鼠心房肌组织胶原纤维沉积和CVF增多以及Collagen I蛋白表达升高。同时，经腺病毒包装系统成功构建差异性表达miR-199及miR-215腺病毒载体，并成功转染至SD大鼠后进行药物诱导心房颤动模型造模。与低表达miR-199比较，高表达miR-199组SD大鼠房颤诱导成功率呈升趋势并促进纤维化，但miR-199差异表达并未明显在房颤中调控Cx40蛋白表达参与电重构。高表达miR-215有促进房颤发生趋势，但miR-215差异表达并未引起Nkx2.5在蛋白水平变化。双荧光素酶报告基因实验结果提示miR-215与Nkx2.5无直接靶向调控关系。我们查阅相关文献，发现NLRP3炎症小体激活可能与房颤机制相关，且在房颤患者中NLRP3激活伴随氧化应激增强和血清Ang II表达水平升高。因而，我们在细胞层面构造了Ang II干预心肌细胞模型，发现miR-223-3p可靶向NLRP3调控Ang II/ROS/NLRP3炎症小体信号通路，抑制细胞焦亡参与房颤。我们同时利用双荧光素酶报告基因实验证实NLRP3是miR-223-3p直接作用的靶基因。因此，miR-223-3p可能是AF治疗中一个新的基因治疗靶点。