LncRNA-p21/HuR/TGFβ调控通路在糖尿病相关心脏纤维化中的作用及机制

Cardiac fibrosis is one of the key pathological changes of the diabetic heart. Previously, we have found that the pro-fibrotic factor TGFβ could promote myofibroblast differentiation and thus fibrosis via cytoplasmic translocation of RNA binding protein HuR, which in turn stabilizes TGFβ, forming a positive feedback loop. Exploring the putative negative regulators of the positive feedback loop would possibly shed light on the prevention and therapy of diabetic cardiomyopathy. Recently, long non-coding RNAs are found to be essential in development and disease. It is revealed that RNA binding protein HuR could bind LncRNA-P21. Our ongoing project has found that LncRNA-P21 expression is reduced in diabetic heart and forced expression of LncRNA-P21 in fibroblast inhibits TGFβ1 induced fibrosis. Taken together, we hypothesize that LncRNA-p21 would possibly block the vicious feedback loop between TGFβ1 and HuR in a ceRNA (competing endogenous RNA) manner, whose drop down might play an essential role in the diabetic cardiac fibrosis. In this study, we will explore the relation among TGFβ, HuR and LncRNA-P21 expression in the control, type 1 and type 2 diabetic heart. Then, we will further analyze the regulatory pathway of LncRNA-p21/HuR/TGFβ and its function and aberration in fibrosis. Through these experiments, we would be possibly characterize the LncRNA-p21/HuR/TGFβ pathway, the regulatory mechanisms and its functional aberration in diabetic cardiac fibrosis, which would shed light on the prevention and therapy of the disease.

课题组前期发现，TGFβ可以活化RNA结合蛋白HuR，后者反馈性增加TGFβ表达;二者形成的正反馈在糖尿病心肌成纤维细胞活化和心脏纤维化中发挥重要作用。寻找该反馈环的内源性抑制因子，对心脏纤维化防治具有重要意义。既往研究表明长链非编码RNA LncRNA-p21可以被HuR识别并结合。我们正在进行的研究发现糖尿病小鼠心脏组织及成纤维细胞中LncRNA-P21表达降低，过表达LncRNA-p21能抑制TGFβ及其诱导的纤维化反应。据此，我们推测，LncRNA-P21可能通过ceRNA（竞争性内源RNA）的方式阻断TGFβ-HuR正反馈，糖尿病条件下LncRNA-P21降低是该病理性反馈环活化并促进纤维化的重要基础。本项目拟建立Ⅰ和Ⅱ型糖尿病小鼠模型，结合细胞学和分子生物学方法，阐明LncRNA-p21/HuR/TGFβ调控通路及其异常在糖尿病心脏纤维化中的作用及分子机制，为其防治提供新思路。

在本青年基金的资助下，我们利用糖尿病模型小鼠探讨了lncRNA-p21和HuR在心脏纤维化中的作用和机制，主要取得以下研究进展：（1）成功建立了HFD饮食诱导的2型糖尿病模型，利用小动物超声发现其心脏功能，特别是舒张功能显著降低，心肌纤维化；（2）发现心脏纤维化是心脏损伤的继发性改变，而过度纤维化是心脏功能，特别是舒张功能下降的重要原因。结合组织和细胞实验，我们证实HuR可以通过稳定TGFβ mRNA稳定性增加TGFβ表达，从而形成正反馈，促进纤维化；HuR转位在心脏成纤维细胞活化和胶原合成中发挥重要作用；（3）发现LncRNA-P21序列中有多个HuR的结合位点（ARE），通过ceRNA方式抑制HuR对TGFβ1的调控；在成纤维细胞过表达lncRNA-p21，TGFβ1和Col1a1的蛋白表达水平均降低，细胞外基质整体水平也降低。（4）探讨了线粒体circRNA在糖尿病心肌纤维化过程中的表达变化。我们通过同源性比对初步描绘出人线粒体circRNA hsa_circ_0089762的小鼠同源物，发现该circRNA在心肌纤维化进程中扮演角色。（5）构建了靶向干预内源HuR的功能的外泌体策略，通过原理验证实验，将RNA结合蛋白HuR与Lamp2b蛋白融合，后者也称为CD107b，编码一种与溶酶体相关的膜糖蛋白。Lamp2b-HuR融合蛋白功能化的外泌体能够将被RNA结合基序识别的RNA靶标驱动至溶酶体以在受体细胞中降解。该系统能够发挥拮抗内源HuR的功能。用Lamp2b-HuR改造的外泌体可能通过溶酶体介导的降解成功降低了RNA靶标的丰度。该外泌体策略，为体内逆转纤维化的研究提供了干预手段，为后续体内干预HuR的功能研究奠定了基础。上述研究共培养研究生2名，申请专利1项，相关论文正在整理中。