LncRNA-GAS5/miR-21/DDAH1途径在糖尿病心肌纤维化中的作用及机制研究

Diabetic cardiomyopathy (DCM), one of the most common microvascular complications, is characterized by cardiac fibrosis. Asymmetrical dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is mainly metabolized by dimethylarginine dimethylaminohydrolase 1 (DDAH1). DDAH1/ADMA pathway plays important roles in the development of diabetic vascular complications. However, whether DDAH1/ADMA pathway is involved in cardiac fibrosis of DCM remains unclear. Non encoding RNAs include small non-encoding RNAs (miRNAs) and long non-encoding RNAs (LncRNAs). LncRNAs and mRNAs modulate each other's abundance by using miRNAs response elements to compete for the binding site of microRNA. It has been reported that miR-21 is elevated in cardiac fibrosis, and siRNA LncRNA-GAS5 increases the levels of miR-21. Bioinformatics analysis shows that DDAH1 may be the target gene of miR-21. Our pilot study showed the decrease in DDAH1 and LncRNA-GAS5, and the increase in miR-21, ADMA and endothelial-mesenchymal transition (EndMT) related protein (such as alpha-SMA and Vimentin) in myocardium of STZ-induced diabetic rat. Based on above results, in the present study we will be planning to explore: the role of cardiac microvascular EndMT in cardiac fibrosis of DCM; the potential role of DDAH1/ADMA pathway in cardiac fibrosis of DCM and the downstream signaling pathway of DDAH1/ADMA in mediating cardiac microvascular EndMT (focusing on TGF-beta1/Smad/Snail or TGF-beta1/non-smad ); the role of LncRNA-GAS5 and miR-21 in cardiac fibrosis of DCM and the potential relationship among DDAH1, LncRNA-GAS5 and miR-21. This study will contribute to the understanding of the pathogenesis of cardiac fibrosis in DCM, and provide novel strategies to seek for the new targets and new drugs responsible for cardiac fibrosis of DCM.

心肌纤维化是糖尿病心肌病（DCM）的重要病理基础。ADMA为NOS抑制物，由DDAH1代谢。DDAH1/ADMA在糖尿病血管病变发展中起重要作用，但是否参与DCM心肌纤维化尚不清楚。LncRNAs和mRNA可通过竞争结合相同miRNA，实现RNA与RNA相互调控。心肌纤维化时miR-21上调，敲低LncRNA-GAS5能上调miR-21，DDAH1可能是miR-21的靶基因。预实验发现，糖尿病大鼠心肌组织DDAH1和LncRNA-GAS5水平下调，miR-21上调，伴随ADMA和内皮细胞间质转化（EndMT）明显。本项目拟探讨：心肌微血管EndMT在DCM心肌纤维化中的作用；DDAH1/ADMA在EndMT和DCM心肌纤维化中的作用及信号途径；LncRNA-GAS5和miR-21在DCM心肌纤维化中的作用及二者的相互关系；LncRNA-GAS5/miR-21调控DDAH1的表达作用及机制。

心肌纤维化是糖尿病心肌病的重要病理基础。我们前期工作证明DDAH/ADMA在糖尿病血管病变中起重要作用，但其是否参与糖尿病心肌纤维化尚不清楚。本项目主要研究内容如下：1) 建立大鼠糖尿病心肌病模型和心肌微血管内皮高糖损伤模型，检测心肌微血管内皮细胞、间质胶原及心肌纤维化情况，心肌微血管内皮间质转化（EndMT）的相关标志蛋白FSP-1、CD31、collagen I等指标，确定DCM心肌纤维化与心肌微血管EndMT的内在联系；2）在糖尿病大鼠模型和心肌微血管内皮高糖损伤模型，结合基因沉默或过表达技术，确定DCM心肌纤维化时DDAH1/ADMA与EndMT的关系；3）在糖尿病大鼠模型和心肌微血管内皮高糖损伤模型，结合免疫共沉淀、基因沉默或表达技术，阐明DCM心肌纤维化时Lnc-GAS5/miR-21与EndMT及DDAH1/ADMA的关系。本项主要发现包括：1）在糖尿病大鼠模型和心肌微血管内皮高糖损伤模型中，发现随着病程的延长，糖尿病组大鼠心肌微血管内皮细胞发生EndMT，确证了高糖环境诱导心肌微血管EndMT，促进心肌纤维化；2）确证了糖尿病心肌病时，DDAH1/ADMA通过TGF-β1途径介导心肌微血管EndMT；3）确证在糖尿病心肌病时，Lnc-DANCR通过上调FoxO1，降低DDAH1表达，促心肌微血管EndMT和心肌纤维化；4））确证在糖尿病心肌病时，下调的GAS5可通过上调miR21水平，抑制DDAH1表达，促进高糖诱导的心肌细胞焦亡，损伤心肌功能。这些发现为糖尿病心肌病心肌纤维化的病理生理机制提供了新的理论依据，为寻找新药靶点，开发抗心肌纤维化药物提供了新思路。