长链非编码RNA调控心肌纤维化的作用与机制

Cardiac fibrosis is the common pathological changes occurred in various cardiac diseases, which can lead to heart failure and arrhythmia. However, the molecular mechanisms remain to be explored. Previously, we found that miR-101/133/590/21 are the critical regulators of cardiac fibrosis. Lately, lncRNAs (Long noncoding RNAs) draw the attention of researchers and are proved to be a new class of regulators in various diseases. Currently, the biological role of lncRNAs in cardiac fibrosis is largely unknown. In our preliminary work we found that the lncRNA expression profiles were significantly altered in fibrotic hearts induced by pressure overload and cardiac infarction, and 67 lncRNAs were predicted to interact with fibrosis-related signaling pathways. Among them, 9 lncRNAs showed expression changes in the same direction in both models. We selected 4 of them for further study. Moreover, we found that MIAT (myocardial infarction associated transcript) participated in the regulation of cardiac fibroblast proliferation and collagen production, which is correlated with the regulation of miR-24 expression. Therefore, we hypothesized that the above 5 lncRNAs are key molecules in regulating cardiac fibrosis, and the mechanisms involve the manipulation of miRNA expression and fibrosis related signaling pathways. In the current project, we will employ RIP, protein mass spectrometry, and genetic manipulated mice model to test the hypothesis at both in vitro and in vivo level in order to confirm the effects of lncRNA on cardiac fibrosis and underlying mechanism.

心肌纤维化是多种心脏疾病的共同病理改变，可导致心力衰竭和心律失常，但其确切分子机制仍有待阐明。我们前期发现非编码RNA miR-101/133/590/21是心肌纤维化的调控分子。长链非编码RNA（lncRNA）是新近引起广泛关注的一类参与重大疾病调节的分子，其与心肌纤维化的关系仍未知。我们发现压力超负荷及缺血纤维化小鼠心肌组织的lncRNA表达谱发生改变，并筛选出与心肌纤维化通路关系密切的67个lncRNAs，且在血管紧张素II刺激的成纤维细胞模型得到验证。两种模型发现9个共性变化的lncRNAs，选择4个作为研究对象。另外，发现MIAT具有抗纤维化作用并与miR-24表达调节相关。因此，我们假设：以上5个lncRNA对心肌纤维化具有调节作用，其机制与调节miRNA及相关纤维化通路有关。我们将应用RIP、蛋白质组学、模式动物等方法验证上述假设，阐明lncRNA调控心肌纤维化的作用与机制。

本研究采用小鼠冠状动脉结扎及主动脉缩窄法建立心肌纤维化模型, 采用基因敲除小鼠、Masson染色、分子克隆、RNA干扰、RNA/蛋白互作、实时定量PCR及基因报告等多种分子生物学技术，从在体、离体及分子水平探讨了lncRNAs（long noncoding RNAs） 对心肌纤维化的调控作用，并探讨其机制。重要发现：1.在小鼠心肌纤维化组织中，lncRNAs表达谱发生改变，变化的lncRNAs同纤维化相关mRNAs存在潜在的调控关系，提示lncRNAs参与了纤维化过程。2. 发现lncRNA MIAT在心脏纤维化组织表达升高，通过miRNA-24/Furin/TGF-β1通路参与心肌纤维化及心脏功能的调节。3. LncRNA PCFL通过作用于miRNA-378/GRB2途径而促进心肌纤维化进程。4. LncRNA-2271在心肌纤维化组织中表达升高，通过与RNA甲基化转移酶METTL3结合而抑制其泛素化，促进心肌纤维化过程。5. 心肌纤维化组织中lncRNA SAIL表达降低，过表达SAIL可抑制人、小鼠心脏成纤维细胞活性和胶原的合成。SAIL通过阻断SAFB和RNA聚合酶Ⅱ的结合，而影响心肌纤维化。6. LncRNA AK081284在糖尿病心肌组织中表达升高，敲减AK081284能抑制高糖诱导的心肌成纤维细胞胶原合成；AK081284参与白细胞介素-17对糖尿病心肌病小鼠心肌纤维化和心脏功能的调控。7. 心力衰竭患者血浆lncRNAs NRON、MHRT水平显著高于非心力衰竭者，NRON、MHRT可作为诊断慢性心力衰竭新的生物标记物。8. LncRNAs CCRR，H19和ACART通过不同机制调控了心律失常、心肌缺血再灌注损伤和心肌细胞凋亡。. 以上结果表明lncRNAs对心脏纤维化、心律失常、心肌缺血等心脏疾病具有重要的调控作用，在不同层面揭示其调控心肌纤维化等心脏疾病的机制。LncRNAs具有成为心力衰竭诊断及预后评价的生物标记物的潜力。以上研究发现扩展和加深了人们对lncRNAs在心脏疾病中的认识，对心脏疾病的基础研究与临床防治具有重要指导意义。本课题相关研究结果共发表SCI收录论文14篇；获省部级科技奖励2项。