心房肌细胞外泌体lncRNA-DLX6-AS1调控房颤心房纤维化机制研究

Exosomal long non-coding RNAs (lncRNAs) mediate cellular communication between cardiomyocytes and fibroblasts via paracrine manner. Our previous studies found that exosomes secreted from tachypacing neonatal atrial myocytes activated atrial fibroblasts and significantly upregulated transforming growth factor-β1 (TGF-β1). Meanwhile, the results of RNA-Seq and bioinformatical analysis indicated that exosomal lncRNA-DLX6-AS1 released from tachypacing atrial myocytes could promote atrial fibrosis via regulating TGFβ1. With the knockdown of DLX6-AS1, the exosomes released from tachypacing atrial myocytes could not activate myofibroblasts and upregulate the expression of TGFβ1. Therefore, we hypothesize that DLX6-AS1 derived from atrial myocytes could upregulate TGFβ1 expression and activate atrial fibroblasts by connecting the miR-26a or the interaction protein, which eventually contributed to atrial fibrosis. The present study mainly focuses on the following three aspects: ①to verify the DLX6-AS1 released from atrial myocytes could enter into and activate atrial fibroblasts; ②to reveal the underlying modulatory mechanisms of exosomal DLX6-AS1 on TGFβ1 expression; ③to elucidate the role of exosomal DLX6-AS1 secreted by atrial myocytes in atrial fibrosis and occurrence of atrial fibrillation in vivo. Our work may provide new insights into the potential role and molecular mechanisms of exosomes in regulating atrial fibrosis.

外泌体可负载lncRNA以旁分泌途径实现心肌细胞与成纤维细胞间信息交流。我们前期研究发现：快速起搏心房肌细胞释放的外泌体可激活心房成纤维细胞，使其TGFβ1表达明显增加；转录组测序和生物信息学预测提示，外泌体lncRNA-DLX6-AS1能够通过调控成纤维细胞TGFβ1表达，引起心房纤维化；沉默DLX6-AS1再起搏的心房肌细胞外泌体不能激活成纤维细胞，且不再使TGFβ1表达上调。我们提出假设：房颤时心房肌细胞源性外泌体DLX6-AS1可能通过结合成纤维细胞内miR-26a或互作蛋白调控TGFβ1表达，促进心房纤维化。本项目拟：①证实心房肌细胞外泌体DLX6-AS1可进入并激活成心房成纤维细胞；②阐明外泌体DLX6-AS1调控心房成纤维细胞TGFβ1表达的分子机制；③在体验证心房肌细胞外泌体DLX6-AS1能够引起心房纤维化，促使房颤发生。本研究将从外泌体环节揭示房颤心房纤维化新机制。

LncRNA通过多种分子机制调节细胞生物学过程。研究证实，lncRNA参与心房颤动（简称房颤）的发生发展。新近报道与我们前期研究均发现，房颤患者心房肌组织及血浆外泌体lncRNA表达存在显著差异，但其在房颤发生中的作用及机制仍不清楚。本项目拟通过细胞水平及临床样本研究证实，房颤时血浆外泌体lncRNA变化情况；探讨上述lncRNA在房颤发生和持续中的作用及相关分子机制。前期外泌体lncRNA测序结果发现，房颤患者与窦性心律者相比，共有42个血浆外泌体lncRNA表达存在显著差异。筛选差异表达倍数＞4的外泌体lncRNA共5个，分别为：DLX6-AS1、MALAT1、NR046944、ESNST00000553637.1和NR003045。对差异显著的5个lncRNA进行qRT-PCR验证。结果显示，与窦性心律组相比，房颤组血浆外泌体DLX6-AS1、MALAT1和NR046944表达水平显著升高，ESNST00000553637.1和NR003045表达水平显著降低。单因素和多因素逻辑回归分析结果表明，血浆外泌体DLX6-AS1、MALAT1、NR046944、ESNST00000553637.1、NR003045及左心房内径与房颤发生独立相关。生物信息学分析显示，血浆外泌体DLX6-AS1、MALAT1、NR046944、ESNST00000553637.1和NR003045可能对细胞内钙离子稳态和炎症等生物学过程具有调控作用。通过细胞水平验证上述lncRNA的功能作用，最后选择NR003045为主要研究对象。结果发现，外源性过表达NR003045能够抑制高频电刺激引起的心房肌细胞凋亡。通过生信分析预测其可能通过调控miR-300引起心房肌细胞凋亡。细胞水平证实，给予miR-300拟似物可导致凋亡相关蛋白Bax、Bcl2、Caspase-9、Caspase-3等显著改变，引起心房肌细胞凋亡，促进心房结构重构。本研究将为今后房颤的防治新靶点提供理论依据。