lncRNA-AK048451增强Smad3促心肌纤维化的机制研究

Myocardial fibrosis is one of the most important leading pathological injury of myocardial remodeling. Our preliminary study did some researches on the effects of AngII/TGF-β1/Smads signal on myocardial fibrosis at the protein level, which was published in "Circ Res". In this year, we found that MicroRNA-21 increased significantly in myocardial fibrosis. MicroRNA-21 down-regulated Smad7 through direct combination and enhanced the increase of collagen caused by TGF-β1, which indicated that RNA was involved in the regulation of Smads. Next, we found that lncRNA-AK048451(lnc-AK) was significantly elevated in the heart tissues of myocardial infarction rat model and the patients with coronary heart disease. lnc-AK is highly conserved between human and rat. The target prediction of LncRNA found Smad3 has mutual binding site with lnc-AK. After down-expressed lnc-AK, it was found that myocardial fibrosis was ameliorated. However, how did lnc-AK mediate myocardial fibrosis? There has no relevant report at present. We assume that lnc-AK enhances the promotion of myocardial fibrosis by up-regulation of Smad3. To confirm the assumption, our project will use the gene chip, adenovirus transfection, ChIP, RNA-pull down, FISH, qRT-PCR, western-blot, etc to obtain direct relation between Smad3 and lnc-AK and find the cellular and molecular mechanism of its promoting myocardial fibrosis, which may help to lay a theoretical foundation for the prevention and treatment of myocardial fibrosis.

心肌纤维化是导致心脏重塑难以逆转的最主要病理改变之一。我们在蛋白水平证实AngII/TGF-β1激活，上调Smad2/3及下调Smad7，促进心肌胶原合成，结果发表在“Circ Res”上。今年我们报道MicroRNA-21直接与Smad7结合并引起其下调，进而增强纤维化，提示RNA参与Smads的调节。本课题前期研究发现lncRNA- AK048451(lnc-AK)在大鼠及人的纤维化心肌中显著升高。下调lnc-AK后，心梗大鼠心肌纤维化明显改善。LncRNA靶点预测IncAK和Smad3有结合位点。我们假设心肌纤维化过程，上调的Inc-AK增加Smad3进而促进纤维化。为证实以上假设，本项目使用基因芯片、腺病毒转染、ChIP、RNA-pull down、FISH、qRT-PCR、Western-blot等方法，旨在获得Inc-AK与Smad3的调控机制，为心肌纤维化的防治奠定理论基础。

心肌纤维化是导致心脏重塑难以逆转的最主要病理改变之一。项目组前期在蛋白水平证实AngII/TGF-β1激活，上调Smad2/3及下调Smad7，促进心肌胶原合成，结果发表在“Circ Res”上。接着我们报道MicroRNA-21直接与Smad7结合并引起其下调，进而增强纤维化，提示RNA参与Smads的调节与心肌纤维化有关。本项目拟在大鼠心肌纤维化模型上，使用基因芯片、质粒转染、荧光素酶报告分析、qRT-PCR、WB等多种实验方法展开研究。课题首先采用人工冠状动脉阻断法建立大鼠心肌梗死模型，观察纤维化心肌中LncRNA的表达，微阵列分析显示心肌梗死后4周，心肌梗死组与假手术组相比有215个lncRNA表达差异，其中Lnc-Ang362上调倍数最高，经qPCR进一步验证。同时，纤维化心肌组织中TGF-β1和胶原纤维I/III表达显著增加，但Smad7的表达显著下调。通过软件预测分析提示Lnc-Ang362与Smad7及miR-200b-3p可能具有结合位点。接着在体外实验，通过上调或及下调Lnc-Ang362的方法，观察Lnc-Ang362对Smad7表达的影响。TGF-β1处理的大鼠心脏成纤维细胞中发现lnc-Ang362表达增加，Smad7表达降低。上调或下调lnc-Ang362的表达可降低或增加Smad7的表达，Smad7表达与胶原纤维I/III型的表达呈负相关。荧光素酶报告分析提示LncRNA-Ang362与Smad7结合并影响其表达。进一步沉默Smad7减弱了lnc-Ang362基因敲除降低成纤维细胞中胶原纤维I/III表达的作用。同时，体外实验构建稳定silnc-Ang362和miR-200b-3p inhibitor，通过影响Lnc-Ang362和miR-200b-3p的表达，发现Lnc-Ang362的上调及miR-200b-3p的下调可引起胶原蛋白的表达增加。构建荧光素酶报告系统进一步验证LncRNA-Ang362与miR-200b-3p可结合并引起miR-200b-3p表达减少。本课题结果提示lnc-Ang362通过抑制Smad7及miR-200b-3p促进心肌梗死后心肌纤维化，lnc-Ang362是一种新的调节心肌梗死后心肌纤维化的lncRNA，为后续进一步深入的心肌纤维化机制研究及寻找防治方法奠定理论基础。